CA2990822A1 - Aminopeptidases for protein hydrlyzates - Google Patents
Aminopeptidases for protein hydrlyzates Download PDFInfo
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- CA2990822A1 CA2990822A1 CA2990822A CA2990822A CA2990822A1 CA 2990822 A1 CA2990822 A1 CA 2990822A1 CA 2990822 A CA2990822 A CA 2990822A CA 2990822 A CA2990822 A CA 2990822A CA 2990822 A1 CA2990822 A1 CA 2990822A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/485—Exopeptidases (3.4.11-3.4.19)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12C1/00—Preparation of malt
- C12C1/027—Germinating
- C12C1/047—Influencing the germination by chemical or physical means
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12C11/00—Fermentation processes for beer
- C12C11/003—Fermentation of beerwort
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12C5/00—Other raw materials for the preparation of beer
- C12C5/004—Enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/11—Aminopeptidases (3.4.11)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
Abstract
The present disclosure provides polypeptides having aminopeptidase activity and isolated nucleic acid sequences encoding the polypeptides. In some embodiments, the disclosure also provides to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the polypeptides. In some embodiments, the present disclosure further provides to methods of obtaining protein hydrolysates useful as flavor improving agents.
Description
TITLE
AMINOPEPTIDASES FOR PROTEIN HYDRLYZATES
CROSS-REFERENCE TO RELATED APPLICATIONS
This applications claims priority to and the benefit of United States provisional patent application numbers 62/185503, filed June 26, 2015; and 62/235937, filed October 1, 2015;
each provisional application titled "NOVEL AMINOPEPTIDASES FOR PROTEIN
HYDROLYSATES".
INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING
The sequence listing provided in the file named "20160610 NB40989-PCT sequence listing prj 5T25.txt" with a size of 192,505 bytes which was created on June 10, 2016 and which is filed herewith, is incorporated by reference herein in its entirety.
BACKGROUND
[0001] Various food and feed products contain protein hydrolysates. This hydrolysis was conventionally accomplished using chemical hydrolysis. However, such chemical hydrolysis led to severe degradation of the amino acids obtained during the hydrolysis, and also to hazardous byproducts formed in the course of the chemical reaction. Increasing concern over the use of protein hydrolysates obtained by chemical hydrolysis has led to the development of enzymatic hydrolysis processes.
AMINOPEPTIDASES FOR PROTEIN HYDRLYZATES
CROSS-REFERENCE TO RELATED APPLICATIONS
This applications claims priority to and the benefit of United States provisional patent application numbers 62/185503, filed June 26, 2015; and 62/235937, filed October 1, 2015;
each provisional application titled "NOVEL AMINOPEPTIDASES FOR PROTEIN
HYDROLYSATES".
INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING
The sequence listing provided in the file named "20160610 NB40989-PCT sequence listing prj 5T25.txt" with a size of 192,505 bytes which was created on June 10, 2016 and which is filed herewith, is incorporated by reference herein in its entirety.
BACKGROUND
[0001] Various food and feed products contain protein hydrolysates. This hydrolysis was conventionally accomplished using chemical hydrolysis. However, such chemical hydrolysis led to severe degradation of the amino acids obtained during the hydrolysis, and also to hazardous byproducts formed in the course of the chemical reaction. Increasing concern over the use of protein hydrolysates obtained by chemical hydrolysis has led to the development of enzymatic hydrolysis processes.
[0002] Enzymatic hydrolysis processes of proteinaceous materials aim at obtaining a high degree of hydrolysis. Polypeptides having aminopeptidase activity catalyze the removal of one or more amino acid residues from the N-terminus of peptides, polypeptides, and proteins. The production of protein hydrolysates with desirable properties and high degrees of hydrolysis generally requires the use of a mixture of peptidase activities. It would be desirable to provide a single component peptidase enzyme which has activity useful for improving the properties and degree of hydrolysis of protein hydrolysates used in food and or feed products either alone or in combination with other enzymes.
[0003] The present disclosure provides novel polypeptides having improved aminopeptidase activity as well as methods for obtaining protein hydrolysates with desirable qualities and high degrees of hydrolysis.
BRIEF DESCRIPTION OF THE DRAWINGS
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0005] Figure 1 shows on the top an example of the overall design of the synthetic genes encoding the pepN 2's enzymes, and at the bottom a close-up of the leader sequence that was used instead of the endogenous secretion signal sequences.
[0006] Figure 2 shows pH dependent glutamic acid liberation of the PepN 2 from Neosartorya fischeri in gluten hydrolyses (no pH-control during hydrolyses).
[0007] Figure 3 shows Temperature dependent glutamic acid liberation of the PepN 2 from Neosartorya fischeri in gluten hydrolyses (no pH-control during hydrolyses).
[0008] Figure 4 shows glutamic acid liberation of different PepN 2.
[0009] Figure 5 shows hydrolysis of WHWLQLKPGQPMY (SEQ ID NO: 26)
[0010] Figure 6 shows hydrolysis of KPGQPMY. (SEQ ID NO: 27)
[0011] Figure 7 shows hydrolysis of QPMY. (SEQ ID NO: 28)
[0012] Figure 8 shows peptide sequences of the substrate as well as cleavage products were typed into Skyline and intensities were calculated in each sample.
[0013] Figure 9 shows the spatial arrangement of six panels Fig. 9A-9F which detail a sequence alignment of several PepN 2 enzymes.
[0014] Figure 10 Hydrolysis of TPAAAR (SEQ ID NO: 29) by TRI032, TRI035, TRI063 (A.
oryzae) as well as COROLASE LAP (fungal exo-peptidase;
oryzae) as well as COROLASE LAP (fungal exo-peptidase;
[0015] DETAILED DESCRIPTION OF THE INVENTION
[0016] The present disclosure provides polypeptides having aminopeptidase activity. The present invention also relates to nucleic acid sequences encoding the polypeptides and to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the polypeptides. The present disclosure also relates to methods for obtaining hydrolysates from proteinaceous substrates which comprise subjecting the proteinaceous material to a polypeptide with aminopeptidase activity alone or in combination with a protease, e.g. an endopeptidase. The present disclosure also relates to methods for obtaining from a proteinaceous substrate a hydrolysate enriched in free glutamic acid and/or peptide bound glutamic acid residues, which methods comprise subjecting the substrate to a polypeptide having aminopeptidase activity. The present disclosure further relates to flavor improving compositions comprising a polypeptide with aminopeptidase activity. The compositions may further comprise additional enzymatic activities.
[0017] In another aspect, the methods described herein may be used in food related applications to improve flavor, such as baking. Alternatively, flavor improvement in foods may be achieved by the addition of hydrolysates obtained by the methods of the invention. In some embodiments, the hydrolysate produced using the aminopeptidases described herein may also have reduced bitterness when compared to an untreated hydrolysate.
[0018] In some embodiments, the invention provides new fungal aminopeptidases (PepNs), their high yield production in a production host (e.g. Trichoderma reesei), and their use for the generation of protein hydrolysates, e.g., for debittering and for glutamate production. As demonstrated in Example 9, aminopeptidases type 2 according to the invention showed improved glutamate release compared with aminopeptidases type 1. Initial results suggest that these PepNs are tolerant to Proline in P1' which is surprising compared to other known aminopeptidases.
Thus, the proline tolerant aminopeptidases taught for use in the present invention are capable of acting on a wide range of peptide and/or protein substrates and due to having such a broad substrate-specificity are not readily inhibited from cleaving substrates enriched in certain amino acids (e.g. proline and/or lysine and/or arginine and/or glycine).
Thus, the proline tolerant aminopeptidases taught for use in the present invention are capable of acting on a wide range of peptide and/or protein substrates and due to having such a broad substrate-specificity are not readily inhibited from cleaving substrates enriched in certain amino acids (e.g. proline and/or lysine and/or arginine and/or glycine).
[0019] The present inventors surprisingly found that aminopeptidases type 2 have better performance that aminopeptidases type 1. Furthermore, the present inventors surprisingly found a group of fungal aminopeptidases type 2 with improved performance. This group of fungal aminopeptidases type 2 has a longer N-terminal in their mature amino acid sequences. Figure 9 shows the amino acid sequence alignment of several aminopeptidases type 2.
[0020] The term "aminopeptidase activity" is defined herein as a peptidase activity which catalyzes the removal of amino acids from the N-terminal end of peptides, oligopeptides or proteins. Defined in a general manner, the aminopeptidase activity is capable of cleaving the amino acid X from the N-terminus of a peptide, polypeptide, or protein, wherein X may represent any amino acid residue selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val, but at least Leu, Glu, Gly, Ala, and/or Pro. It will be understood that the polypeptides having aminopeptidase activity of the present invention may be unspecific as to the amino acid to be cleaved of the N-terminus of the peptide, polypeptide substrate.
[0021] In some embodiments, the present invention relates to an isolated polypeptide having aminopeptidase activity which has a predicted mature sequence with more than 13 residues on the N-terminal side of the conserved residue I/V in position 67 as shown in the sequence alignment of Figure 9, or a fragment thereof, wherein the fragment has aminopeptidase activity.
[0022] In some embodiments, the present invention relates to isolated polypeptides having an amino acid sequence which has a degree of identity to the mature amino acid sequence of SEQ
ID NO: 1 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have aminopeptidase activity (hereinafter "homologous polypeptides"). In some embodiments, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID
NO: 1. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 1, the mature amino sequence or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 1. In another embodiment, the polypeptide of the present invention has the amino acid sequence of SEQ ID NO:
1 or a fragment thereof, wherein the fragment has aminopeptidase activity. A fragment of SEQ
ID NO: 1 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. In some embodiments the fragment comprises the sequence of SEQ ID
NO:18. In some embodiments, the polypeptide has the amino acid sequence of SEQ
ID NO: 1.
ID NO: 1 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have aminopeptidase activity (hereinafter "homologous polypeptides"). In some embodiments, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID
NO: 1. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 1, the mature amino sequence or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 1. In another embodiment, the polypeptide of the present invention has the amino acid sequence of SEQ ID NO:
1 or a fragment thereof, wherein the fragment has aminopeptidase activity. A fragment of SEQ
ID NO: 1 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. In some embodiments the fragment comprises the sequence of SEQ ID
NO:18. In some embodiments, the polypeptide has the amino acid sequence of SEQ
ID NO: 1.
[0023] In some embodiments, the present invention relates to isolated polypeptides having an amino acid sequence which has a degree of identity to the mature amino acid sequence of SEQ
ID NO: 2 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have aminopeptidase activity (hereinafter "homologous polypeptides"). In some embodiments, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID
NO: 2. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 2, the mature amino sequence or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 2. In another embodiment, the polypeptide of the present invention has the amino acid sequence of SEQ ID NO:
2 or a fragment thereof, wherein the fragment has aminopeptidase activity. A fragment of SEQ
ID NO: 2 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. In some embodiments the fragment comprises the sequence of SEQ ID
NO:19. In some embodiments, the polypeptide has the amino acid sequence of SEQ
ID NO: 2.
ID NO: 2 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have aminopeptidase activity (hereinafter "homologous polypeptides"). In some embodiments, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID
NO: 2. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 2, the mature amino sequence or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 2. In another embodiment, the polypeptide of the present invention has the amino acid sequence of SEQ ID NO:
2 or a fragment thereof, wherein the fragment has aminopeptidase activity. A fragment of SEQ
ID NO: 2 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. In some embodiments the fragment comprises the sequence of SEQ ID
NO:19. In some embodiments, the polypeptide has the amino acid sequence of SEQ
ID NO: 2.
[0024] In some embodiments, the present invention relates to isolated polypeptides having an amino acid sequence which has a degree of identity to the mature amino acid sequence of SEQ
ID NO: 3 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have aminopeptidase activity (hereinafter "homologous polypeptides"). In some embodiments, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID
NO: 3. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 3, the mature amino sequence or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 3. In another embodiment, the polypeptide of the present invention has the amino acid sequence of SEQ ID NO:
3 or a fragment thereof, wherein the fragment has aminopeptidase activity. A fragment of SEQ
ID NO: 3 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. In some embodiments the fragment comprises the sequence of SEQ ID
NO:20. In some embodiments, the polypeptide has the amino acid sequence of SEQ
ID NO: 3.
ID NO: 3 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have aminopeptidase activity (hereinafter "homologous polypeptides"). In some embodiments, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID
NO: 3. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 3, the mature amino sequence or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 3. In another embodiment, the polypeptide of the present invention has the amino acid sequence of SEQ ID NO:
3 or a fragment thereof, wherein the fragment has aminopeptidase activity. A fragment of SEQ
ID NO: 3 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. In some embodiments the fragment comprises the sequence of SEQ ID
NO:20. In some embodiments, the polypeptide has the amino acid sequence of SEQ
ID NO: 3.
[0025] In some embodiments, the present invention relates to isolated polypeptides having an amino acid sequence which has a degree of identity to the mature amino acid sequence of SEQ
ID NO: 4 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have aminopeptidase activity (hereinafter "homologous polypeptides"). In some embodiments, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID
NO: 4. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 4, the mature amino sequence or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 4. In another embodiment, the polypeptide of the present invention has the amino acid sequence of SEQ ID NO:
4 or a fragment thereof, wherein the fragment has aminopeptidase activity. A fragment of SEQ
ID NO: 4 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. In some embodiments the fragment comprises the sequence of SEQ ID
NO:21. In some embodiments, the polypeptide has the amino acid sequence of SEQ
ID NO: 4.
ID NO: 4 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have aminopeptidase activity (hereinafter "homologous polypeptides"). In some embodiments, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID
NO: 4. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 4, the mature amino sequence or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 4. In another embodiment, the polypeptide of the present invention has the amino acid sequence of SEQ ID NO:
4 or a fragment thereof, wherein the fragment has aminopeptidase activity. A fragment of SEQ
ID NO: 4 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. In some embodiments the fragment comprises the sequence of SEQ ID
NO:21. In some embodiments, the polypeptide has the amino acid sequence of SEQ
ID NO: 4.
[0026] In some embodiments, the present invention relates to isolated polypeptides having an amino acid sequence which has a degree of identity to the mature amino acid sequence of SEQ
ID NO: 5 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have aminopeptidase activity (hereinafter "homologous polypeptides"). In some embodiments, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID
NO: 5. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 5, the mature amino sequence or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 5. In another embodiment, the polypeptide of the present invention has the amino acid sequence of SEQ ID NO:
5 or a fragment thereof, wherein the fragment has aminopeptidase activity. A fragment of SEQ
ID NO: 5 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. In some embodiments the fragment comprises the sequence of SEQ ID
NO:22. In some embodiments, the polypeptide has the amino acid sequence of SEQ
ID NO: 5.
ID NO: 5 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have aminopeptidase activity (hereinafter "homologous polypeptides"). In some embodiments, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID
NO: 5. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 5, the mature amino sequence or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 5. In another embodiment, the polypeptide of the present invention has the amino acid sequence of SEQ ID NO:
5 or a fragment thereof, wherein the fragment has aminopeptidase activity. A fragment of SEQ
ID NO: 5 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. In some embodiments the fragment comprises the sequence of SEQ ID
NO:22. In some embodiments, the polypeptide has the amino acid sequence of SEQ
ID NO: 5.
[0027] In some embodiments, the present invention relates to isolated polypeptides having an amino acid sequence which has a degree of identity to the mature amino acid sequence of SEQ
ID NO: 6 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have aminopeptidase activity (hereinafter "homologous polypeptides"). In some embodiments, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID
NO: 6. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 6, the mature amino sequence or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 6. In another embodiment, the polypeptide of the present invention has the amino acid sequence of SEQ ID NO:
6 or a fragment thereof, wherein the fragment has aminopeptidase activity. A fragment of SEQ
ID NO: 6 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. In some embodiments the fragment comprises the sequence of SEQ ID
NO:23. In some embodiments, the polypeptide has the amino acid sequence of SEQ
ID NO: 6.
ID NO: 6 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have aminopeptidase activity (hereinafter "homologous polypeptides"). In some embodiments, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID
NO: 6. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 6, the mature amino sequence or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 6. In another embodiment, the polypeptide of the present invention has the amino acid sequence of SEQ ID NO:
6 or a fragment thereof, wherein the fragment has aminopeptidase activity. A fragment of SEQ
ID NO: 6 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. In some embodiments the fragment comprises the sequence of SEQ ID
NO:23. In some embodiments, the polypeptide has the amino acid sequence of SEQ
ID NO: 6.
[0028] In some embodiments, the present invention relates to isolated polypeptides having an amino acid sequence which has a degree of identity to the mature amino acid sequence of SEQ
ID NO: 7 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have aminopeptidase activity (hereinafter "homologous polypeptides"). In some embodiments, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID
NO: 7. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 7, the mature amino sequence or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 7. In another embodiment, the polypeptide of the present invention has the amino acid sequence of SEQ ID NO:
7 or a fragment thereof, wherein the fragment has aminopeptidase activity. A fragment of SEQ
ID NO: 7 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. In some embodiments the fragment comprises the sequence of SEQ ID
NO:24. In some embodiments, the polypeptide has the amino acid sequence of SEQ
ID NO: 7.
ID NO: 7 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have aminopeptidase activity (hereinafter "homologous polypeptides"). In some embodiments, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID
NO: 7. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 7, the mature amino sequence or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 7. In another embodiment, the polypeptide of the present invention has the amino acid sequence of SEQ ID NO:
7 or a fragment thereof, wherein the fragment has aminopeptidase activity. A fragment of SEQ
ID NO: 7 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. In some embodiments the fragment comprises the sequence of SEQ ID
NO:24. In some embodiments, the polypeptide has the amino acid sequence of SEQ
ID NO: 7.
[0029] In some embodiments, the present invention relates to isolated polypeptides having an amino acid sequence which has a degree of identity to the mature amino acid sequence of SEQ
ID NO: 8 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have aminopeptidase activity (hereinafter "homologous polypeptides"). In some embodiments, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID
NO: 8. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 8, the mature amino sequence or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 8. In another embodiment, the polypeptide of the present invention has the amino acid sequence of SEQ ID NO:
8 or a fragment thereof, wherein the fragment has aminopeptidase activity. A fragment of SEQ
ID NO: 8 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. In some embodiments the fragment comprises the sequence of SEQ ID
NO:25. In some embodiments, the polypeptide has the amino acid sequence of SEQ
ID NO: 8.
ID NO: 8 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have aminopeptidase activity (hereinafter "homologous polypeptides"). In some embodiments, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID
NO: 8. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 8, the mature amino sequence or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 8. In another embodiment, the polypeptide of the present invention has the amino acid sequence of SEQ ID NO:
8 or a fragment thereof, wherein the fragment has aminopeptidase activity. A fragment of SEQ
ID NO: 8 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. In some embodiments the fragment comprises the sequence of SEQ ID
NO:25. In some embodiments, the polypeptide has the amino acid sequence of SEQ
ID NO: 8.
[0030] Preferably, a fragment contains at least 330 amino acid residues, more preferably at least 380 amino acid residues, and most preferably at least 430 amino acid residues.
[0031] An allelic variant denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations.
[0032] The "parent enzyme" as used herein is an aminopeptidase enzyme which has all of the amino acid residues of the polypeptide as shown in SEQ ID NO: 1, or SEQ ID NO:
2, or SEQ ID
NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ
ID NO:
8. In this respect, and for example, the parent will have all of the modifications (which may be zero, one or more than one depending on the variant) of the variant polypeptide.
2, or SEQ ID
NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ
ID NO:
8. In this respect, and for example, the parent will have all of the modifications (which may be zero, one or more than one depending on the variant) of the variant polypeptide.
[0033] In some embodiments, the present invention relates to isolated polypeptides having aminopeptidase activity which are encoded by nucleic acid sequences which hybridize under low stringency conditions, more preferably medium stringency conditions, and most preferably high stringency conditions, with an oligonucleotide probe which hybridizes under the same conditions with the polypeptide encoding part of nucleic acid sequence of SEQ ID NO: 9, or SEQ ID NO:
10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO:
15, or SEQ ID NO: 16 or its complementary strand (J. Sambrook, E.F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York); or allelic variants and fragments of the polypeptides, wherein the fragments have aminopeptidase activity.
10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO:
15, or SEQ ID NO: 16 or its complementary strand (J. Sambrook, E.F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York); or allelic variants and fragments of the polypeptides, wherein the fragments have aminopeptidase activity.
[0034] Hybridization indicates that the nucleic acid sequence hybridizes to the oligonucleotide probe corresponding to the polypeptide encoding part of the nucleic acid sequence shown in SEQ
ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO:
13, SEQ
ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16, under low to high stringency conditions (i.e., prehybridization and hybridization at 42 C in 5X SSPE, 0.3% SDS, 200 Pg/ml sheared and denatured salmon sperm DNA, and either 25, 35 or 50% formamide for low, medium and high stringencies, respectively), following standard Southern blotting procedures.
ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO:
13, SEQ
ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16, under low to high stringency conditions (i.e., prehybridization and hybridization at 42 C in 5X SSPE, 0.3% SDS, 200 Pg/ml sheared and denatured salmon sperm DNA, and either 25, 35 or 50% formamide for low, medium and high stringencies, respectively), following standard Southern blotting procedures.
[0035] The amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO:
3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or a partial sequence thereof may be used to design an oligonucleotide probe, or a nucleic acid sequence encoding a polypeptide of the present invention, such as the polypeptide encoding part of nucleic acid sequence of SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID
NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16, or a subsequence thereof, may be used to identify and clone DNA encoding polypeptides having aminopeptidase activity from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, preferably at least 25, and more preferably at least 40 nucleotides in length. Longer probes can also be used.
Both DNA and RNA
probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin).
3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or a partial sequence thereof may be used to design an oligonucleotide probe, or a nucleic acid sequence encoding a polypeptide of the present invention, such as the polypeptide encoding part of nucleic acid sequence of SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID
NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16, or a subsequence thereof, may be used to identify and clone DNA encoding polypeptides having aminopeptidase activity from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, preferably at least 25, and more preferably at least 40 nucleotides in length. Longer probes can also be used.
Both DNA and RNA
probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin).
[0036] Thus, a genomic, cDNA or combinatorial chemical library prepared from such other organisms may be screened for DNA which hybridizes with the probes described above and which encodes a polypeptide having aminopeptidase activity. Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA which is homologous with the polypeptide encoding part of nucleic acid sequence of SEQ
ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO:
13, SEQ
ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16, the carrier material is used in a Southern blot in which the carrier material is finally washed three times for 30 minutes each using 2 x SSC, 0.2% SDS preferably at least 50 C, more preferably at least 55 C, more preferably at least 60 C, more preferably at least 65 C, even more preferably at least 70 C, and most preferably at least 75 C. Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using X-ray film.
ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO:
13, SEQ
ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16, the carrier material is used in a Southern blot in which the carrier material is finally washed three times for 30 minutes each using 2 x SSC, 0.2% SDS preferably at least 50 C, more preferably at least 55 C, more preferably at least 60 C, more preferably at least 65 C, even more preferably at least 70 C, and most preferably at least 75 C. Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using X-ray film.
[0037] The terms "modifying" and "modification" as used herein refers to a substitution when compared with the wild-type aminopeptidase polypeptide sequence of the closest identity. The comparison is made by aligning both the variant aminopeptidase polypeptide and the wild-type aminopeptidase with the reference sequence shown as SEQ ID NO: 1, or SEQ ID
NO: 2, or SEQ
ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID
NO: 8.
NO: 2, or SEQ
ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID
NO: 8.
[0038] Likewise, "equivalent modification" as used herein refers to carrying out the same modification (usually a substitution) to an amino acid which is at an equivalent position in another aminopeptidase.
[0039] In one aspect, the aminopeptidase sequence according to the present invention is in an isolated form. The term "isolated" means that the aminopeptidase sequence is at least substantially free from at least one other component with which the aminopeptidase sequence is naturally associated in nature and as found in nature. The aminopeptidase sequence of the present invention may be provided in a form that is substantially free of one or more contaminants with which the substance might otherwise be associated. Thus, for example it may be substantially free of one or more potentially contaminating polypeptides and/or nucleic acid molecules.
[0040] In one aspect, the aminopeptidase sequence according to the present invention is in a purified form. The term "purified" means that a given component is present at a high level. The component is desirably the predominant component present in a composition.
Preferably, the aminopeptidase is present at a level of at least about 90%, or at least about 95% or at least about 98%, said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration.
Preferably, the aminopeptidase is present at a level of at least about 90%, or at least about 95% or at least about 98%, said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration.
[0041] As used herein, the singular "a," "an" and "the" includes the plural unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acid sequences are written left to right in 5' to 3' orientation; and amino acid sequences are written left to right in amino to carboxy orientation. It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described herein, absent an indication to the contrary.
[0042] Terms and abbreviations not defined should be accorded their ordinary meaning as used in the art. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
Unless otherwise indicated, the practice of the present disclosure involves conventional techniques commonly used in molecular biology, protein engineering, and microbiology. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present disclosure, some suitable methods and materials are described herein. The terms defined immediately below are more fully described by reference to the Specification as a whole.
Nucleotide sequence
Unless otherwise indicated, the practice of the present disclosure involves conventional techniques commonly used in molecular biology, protein engineering, and microbiology. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present disclosure, some suitable methods and materials are described herein. The terms defined immediately below are more fully described by reference to the Specification as a whole.
Nucleotide sequence
[0043] The scope of the present invention encompasses nucleotide sequences encoding aminopeptidases having the specific properties as defined herein.
[0044] In some embodiments, the nucleic acid sequence encodes a polypeptide obtained from Aspergillus, e.g., Aspergillus clavatus. In some embodiments, the nucleic acid sequence encodes a polypeptide obtained from Neosartorya, e.g. Neosartorya fischeri.
[0045] In some embodiments, the present invention relates to a nucleic acid encoding a polypeptide having aminopeptidase activity which has a predicted mature sequence with more than 13 residues on the N-terminal side of the conserved residue I/V in position 67 as shown in the sequence alignment of Figure 9, or a fragment thereof, wherein the fragment has aminopeptidase activity.
[0046] In some embodiments, the polynucleotide of the present invention is a polynucleotide having a specified degree of nucleic acid homology to the exemplified polynucleotide. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 50, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the polypeptide encoding part of nucleic acid sequence of SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO:
11, or SEQ
ID NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO:
16. In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ
ID NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16. In other embodiments, the polynucleotide of the present invention may also have a complementary nucleic acid sequence to a nucleic acid sequence selected from the group consisting of SEQ ID
NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID
NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a recombinant polypeptide or an active fragment thereof, comprising an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identity to the amino acid sequence selected from the group consisting of SEQ ID NO:
1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID
NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a recombinant polypeptide or an active fragment thereof, comprising an amino acid sequence selected from the group consisting of SEQ ID
NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ
ID NO: 7, or SEQ ID NO: 8. Homology can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.
11, or SEQ
ID NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO:
16. In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ
ID NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16. In other embodiments, the polynucleotide of the present invention may also have a complementary nucleic acid sequence to a nucleic acid sequence selected from the group consisting of SEQ ID
NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID
NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a recombinant polypeptide or an active fragment thereof, comprising an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identity to the amino acid sequence selected from the group consisting of SEQ ID NO:
1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID
NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a recombinant polypeptide or an active fragment thereof, comprising an amino acid sequence selected from the group consisting of SEQ ID
NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ
ID NO: 7, or SEQ ID NO: 8. Homology can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.
[0047] The present invention also encompasses nucleic acid sequences which encode a polypeptide having the amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID
NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ
ID NO:
8, which differ from SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID
NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ
ID NO: 9, or SEQ
ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID NO:
14, or SEQ
ID NO: 15, or SEQ ID NO: 16 which encode fragments of SEQ ID NO: 1, or SEQ ID
NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ
ID NO: 8 which have aminopeptidase activity. A subsequence of SEQ ID NO: 9, or SEQ ID
NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID
NO: 15, or SEQ ID NO: 16 is a nucleic acid sequence encompassed by SEQ ID NO:
9, or SEQ
ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID NO:
14, or SEQ
ID NO: 15, or SEQ ID NO: 16 except that one or more nucleotides from the 5' end and/or 3' end have been deleted. Preferably, a subsequence contains at least 990 nucleotides, more preferably at least 1140 nucleotides, and most preferably at least 1290 nucleotides.
NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ
ID NO:
8, which differ from SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID
NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ
ID NO: 9, or SEQ
ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID NO:
14, or SEQ
ID NO: 15, or SEQ ID NO: 16 which encode fragments of SEQ ID NO: 1, or SEQ ID
NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ
ID NO: 8 which have aminopeptidase activity. A subsequence of SEQ ID NO: 9, or SEQ ID
NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID
NO: 15, or SEQ ID NO: 16 is a nucleic acid sequence encompassed by SEQ ID NO:
9, or SEQ
ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID NO:
14, or SEQ
ID NO: 15, or SEQ ID NO: 16 except that one or more nucleotides from the 5' end and/or 3' end have been deleted. Preferably, a subsequence contains at least 990 nucleotides, more preferably at least 1140 nucleotides, and most preferably at least 1290 nucleotides.
[0048] The term "nucleotide sequence" as used herein refers to an oligonucleotide sequence or polynucleotide sequence, and variant, homologues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be double-stranded or single-stranded whether representing the sense or anti-sense strand.
[0049] The term "nucleotide sequence" in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA
sequence coding for the present invention.
sequence coding for the present invention.
[0050] In a preferred embodiment, the nucleotide sequence when relating to and when encompassed by theper se scope of the present invention does not include the native nucleotide sequence according to the present invention when in its natural environment and when it is linked to its naturally associated sequence(s) that is/are also in its/their natural environment. For ease of reference, we shall call this preferred embodiment the "non-native nucleotide sequence". In this regard, the term "native nucleotide sequence" means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment. However, the amino acid sequence encompassed by scope the present invention can be isolated and/or purified post expression of a nucleotide sequence in its native organism. Preferably, however, the amino acid sequence encompassed by scope of the present invention may be expressed by a nucleotide sequence in its native organism but wherein the nucleotide sequence is not under the control of the promoter with which it is naturally associated within that organism.
[0051] Typically, the nucleotide sequence encompassed by the scope of the present invention is prepared using recombinant DNA techniques (i.e. recombinant DNA). However, in an alternative embodiment of the invention, the nucleotide sequence could be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers MH et at., (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al., (1980) Nuc Acids Res Symp Ser 225-232).
[0052] The present invention also relates to nucleic acid sequences which have a degree of homology to the nucleic acid sequence of SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO:
11, or SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO:
16of at least about 50%, preferably about 60%, preferably about 70%, preferably about 80%, more preferably about 90%, even more preferably about 95%, and most preferably about 97%
homology, which encode an active polypeptide. For purposes of the present invention, the degree of homology between two nucleic acid sequences is determined by the CLUSTAL
method (Higgins, 1989, supra) with an identity table, a gap penalty of 10, and a gap length penalty of 10.
11, or SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO:
16of at least about 50%, preferably about 60%, preferably about 70%, preferably about 80%, more preferably about 90%, even more preferably about 95%, and most preferably about 97%
homology, which encode an active polypeptide. For purposes of the present invention, the degree of homology between two nucleic acid sequences is determined by the CLUSTAL
method (Higgins, 1989, supra) with an identity table, a gap penalty of 10, and a gap length penalty of 10.
[0053] Modification of a nucleic acid sequence encoding a polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term "substantially similar" to the polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the polypeptide isolated from its native source. For example, it may be of interest to synthesize variants of the polypeptide where the variants differ in specific activity, thermostability, pH optimum, or the like using, e.g., site-directed mutagenesis. The analogous sequence may be constructed on the basis of the nucleic acid sequence presented as the polypeptide encoding part of nucleic acid sequence of SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO:
12, or SEQ
ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide encoded by the nucleic acid sequence, but which corresponds to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.
12, or SEQ
ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide encoded by the nucleic acid sequence, but which corresponds to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.
[0054] It will be apparent to those skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active polypeptide.
Amino acid residues essential to the activity of the polypeptide encoded by the isolated nucleic acid sequence of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for aminopeptidase activity to identify amino acid residues that are critical to the activity of the molecule. Sites of substrate-enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labelling (see, e.g., de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).
Compositions
Amino acid residues essential to the activity of the polypeptide encoded by the isolated nucleic acid sequence of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for aminopeptidase activity to identify amino acid residues that are critical to the activity of the molecule. Sites of substrate-enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labelling (see, e.g., de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).
Compositions
[0055] In one aspect, the present invention also relates to compositions comprising aminopeptidases and amino acid sequences and/or nucleotide sequences as described herein.
[0056] In some embodiments, the present invention provides compositions comprising a polypeptide having aminopeptidase activity which has a predicted mature sequence with more than 13 residues on the N-terminal side of the conserved residue I/V in position 67 as shown in the sequence alignment of Figure 9, or a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the compositions comprise polypeptides having at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97% homology to a polypeptide having aminopeptidase activity which has a predicted mature sequence with more than 13 residues on the N-terminal side of the conserved residue IN in position 67 as shown in the sequence alignment of Figure 9.
[0057] In some embodiments, the present invention provides a general composition comprising at least one aminopeptidase having an amino acid sequence which has a degree of identity to the amino acid sequence of SEQ ID NO: 1 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%. In some embodiments, the compositions comprise homologous polypeptides having an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID NO: 1. In some embodiments, the compositions comprise the amino acid sequence of SEQ ID NO: 1 or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the compositions comprise the amino acid sequence of SEQ ID NO: 1. In another embodiment, the compositions comprise an aminopeptidase having the amino acid sequence of SEQ ID NO: lor a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the compositions comprise a polypeptide having the amino acid sequence of SEQ ID
NO: 1
NO: 1
[0058] In some embodiments, the present invention provides a general composition comprising at least one aminopeptidase having an amino acid sequence which has a degree of identity to the amino acid sequence of SEQ ID NO: 2 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%. In some embodiments, the compositions comprise homologous polypeptides having an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID NO: 2. In some embodiments, the compositions comprise the amino acid sequence of SEQ ID NO: 2 or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the compositions comprise the amino acid sequence of SEQ ID NO: 2. In another embodiment, the compositions comprise an aminopeptidase having the amino acid sequence of SEQ ID NO: 2 or a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the compositions comprise a polypeptide having the amino acid sequence of SEQ ID
NO: 2.
NO: 2.
[0059] In some embodiments, the present invention provides a general composition comprising at least one aminopeptidase having an amino acid sequence which has a degree of identity to the amino acid sequence of SEQ ID NO: 3 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%. In some embodiments, the compositions comprise homologous polypeptides having an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID NO: 3. In some embodiments, the compositions comprise the amino acid sequence of SEQ ID NO: 3 or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the compositions comprise the amino acid sequence of SEQ ID NO: 3. In another embodiment, the compositions comprise an aminopeptidase having the amino acid sequence of SEQ ID NO: 3 or a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the compositions comprise a polypeptide having the amino acid sequence of SEQ ID
NO: 3.
NO: 3.
[0060] In some embodiments, the present invention provides a general composition comprising at least one aminopeptidase having an amino acid sequence which has a degree of identity to the amino acid sequence of SEQ ID NO: 4 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%. In some embodiments, the compositions comprise homologous polypeptides having an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID NO: 4. In some embodiments, the compositions comprise the amino acid sequence of SEQ ID NO: 4 or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the compositions comprise the amino acid sequence of SEQ ID NO: 4. In another embodiment, the compositions comprise an aminopeptidase having the amino acid sequence of SEQ ID NO: 4 or a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the compositions comprise a polypeptide having the amino acid sequence of SEQ ID
NO: 4.
NO: 4.
[0061] In some embodiments, the present invention provides a general composition comprising at least one aminopeptidase having an amino acid sequence which has a degree of identity to the amino acid sequence of SEQ ID NO: 5 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%. In some embodiments, the compositions comprise homologous polypeptides having an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID NO: 5. In some embodiments, the compositions comprise the amino acid sequence of SEQ ID NO: 5 or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the compositions comprise the amino acid sequence of SEQ ID NO: 5. In another embodiment, the compositions comprise an aminopeptidase having the amino acid sequence of SEQ ID NO: 5 or a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the compositions comprise a polypeptide having the amino acid sequence of SEQ ID
NO: 5.
NO: 5.
[0062] In some embodiments, the present invention provides a general composition comprising at least one aminopeptidase having an amino acid sequence which has a degree of identity to the amino acid sequence of SEQ ID NO: 6 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%. In some embodiments, the compositions comprise homologous polypeptides having an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID NO: 6. In some embodiments, the compositions comprise the amino acid sequence of SEQ ID NO: 6 or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the compositions comprise the amino acid sequence of SEQ ID NO: 6. In another embodiment, the compositions comprise an aminopeptidase having the amino acid sequence of SEQ ID NO: 6 or a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the compositions comprise a polypeptide having the amino acid sequence of SEQ ID
NO: 6.
NO: 6.
[0063] In some embodiments, the present invention provides a general composition comprising at least one aminopeptidase having an amino acid sequence which has a degree of identity to the amino acid sequence of SEQ ID NO: 7 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%. In some embodiments, the compositions comprise homologous polypeptides having an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID NO: 7. In some embodiments, the compositions comprise the amino acid sequence of SEQ ID NO: 7 or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the compositions comprise the amino acid sequence of SEQ ID NO: 7. In another embodiment, the compositions comprise an aminopeptidase having the amino acid sequence of SEQ ID NO: 7 or a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the compositions comprise a polypeptide having the amino acid sequence of SEQ ID
NO: 7.
NO: 7.
[0064] In some embodiments, the present invention provides a general composition comprising at least one aminopeptidase having an amino acid sequence which has a degree of identity to the amino acid sequence of SEQ ID NO: 8 of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%. In some embodiments, the compositions comprise homologous polypeptides having an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID NO: 8. In some embodiments, the compositions comprise the amino acid sequence of SEQ ID NO: 8 or an allelic variant; and a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the compositions comprise the amino acid sequence of SEQ ID NO: 8. In another embodiment, the compositions comprise an aminopeptidase having the amino acid sequence of SEQ ID NO: 8 or a fragment thereof, wherein the fragment has aminopeptidase activity. In some embodiments, the compositions comprise a polypeptide having the amino acid sequence of SEQ ID
NO: 8.
NO: 8.
[0065] In some embodiments, the compositions comprise a polynucleotide having a specified degree of nucleic acid homology to the exemplified polynucleotide. In some embodiments, the compositions comprise a polynucleotide comprising a nucleic acid sequence having at least 50, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%
identity to the nucleic acid sequence of the polypeptide encoding part of nucleic acid sequence of SEQ ID
NO: 9, or SEQ ID
NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID
NO: 15, or SEQ ID NO: 16. In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID
NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID
NO: 16 In other embodiments, the compositions comprise a polynucleotide with a complementary nucleic acid sequence to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO:
12, or SEQ
ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16. In some embodiments, the compositions comprise a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide or an active fragment thereof, comprising an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID
NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8.
In some embodiments, the compositions comprise a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide or an active fragment thereof, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID
NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ
ID NO:
8.
identity to the nucleic acid sequence of the polypeptide encoding part of nucleic acid sequence of SEQ ID
NO: 9, or SEQ ID
NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID
NO: 15, or SEQ ID NO: 16. In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID
NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID
NO: 16 In other embodiments, the compositions comprise a polynucleotide with a complementary nucleic acid sequence to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO:
12, or SEQ
ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16. In some embodiments, the compositions comprise a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide or an active fragment thereof, comprising an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID
NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8.
In some embodiments, the compositions comprise a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide or an active fragment thereof, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID
NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ
ID NO:
8.
[0066] In some embodiments, the composition may comprise multiple enzymatic activities, such as an aminopeptidase, an amylase, a carbohydrase, a carboxypeptidase, a catalase, a cellulase, a chitinase, a cutinase, a cyclodextrin glycosyltransferase, a deoxyribonuclease, an esterase, an alpha-galactosidase, a beta-galactosidase, a glucoamylase, an alpha-glucosidase, a beta-glucosidase, a haloperoxidase, an invertase, a laccase, a lipase, a mannosidase, an oxidase, a pectinolytic enzyme, a peptidoglutaminase, a peroxidase, a phytase, a polyphenoloxidase, a proteolytic enzyme, a ribonuclease, a transglutaminase, or a xylanase. The additional enzyme(s) may be producible by means of a microorganism belonging to the genus Aspergillus, preferably Aspergillus aculeatus, Aspergillus awamori, Aspergillus niger, or Aspergillus oryzae, or Trichoderma, Hum/cola, preferably Hum/cola insolens, or Fusarium, preferably Fusarium bactridioides, Fusarium cereal/s, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenaium.
[0067] In some embodiments, the invention relates to compositions comprising a polypeptide with aminopeptidase activity and a suitable carrier. Any suitable carrier known in the art may be used including those described herein. In another embodiment, the compositions further comprise an endopeptidase. In some embodiments, the compositions further comprise one or more unspecific-acting endo- and/or exo-peptidase enzymes. In some embodiments, the compositions further comprise one or more specific-acting endo- and/or exo-peptidase enzymes.
[0068] In some embodiments, the specific acting proteolytic enzyme is an endopeptidase such as a glutamylendopeptidase (EC 3.4.21.19); a lysyl endopeptidase (EC 3.4.21.50);
a leucyl endopeptidase (EC 3.4.21.57); a glycyl endopeptidase (EC 3.4.22.25); a prolyl endopeptidase (EC 3.4.21.26); trypsin (EC 3.4.21.4) or a trypsin-like (lysine/arginine specific) endopeptidase;
or a peptidyl-Asp metalloendopeptidase (EC 3.4.24.33).
a leucyl endopeptidase (EC 3.4.21.57); a glycyl endopeptidase (EC 3.4.22.25); a prolyl endopeptidase (EC 3.4.21.26); trypsin (EC 3.4.21.4) or a trypsin-like (lysine/arginine specific) endopeptidase;
or a peptidyl-Asp metalloendopeptidase (EC 3.4.24.33).
[0069] In some embodiments, the exopeptidase enzyme is selected from the group consisting of tripeptidyl aminopeptidase, dipeptidyl aminopeptidase, carboxypeptidase and other aminopeptidases.
[0070] In some embodiments, the one or more endo- and/or exo-peptidase enzymes are selected from the group consisting of acid fungal endopeptidase, metallo neutral endopeptidase, alkaline serine endopeptidase, subtilisin, bromelain, thermostable bacterial neutral endopeptidase, alkaline serine endopeptidase.
[0071] In some embodiments, the endo- and/or exo-peptidase enzymes for use in the present invention may be one or more of the proteases in one or more of the commercial products below:
Commercial product ., Company Protease type Protease source ..
ALPHALASE AFP Genencor/DuPont Acid fungal Trichoderma reesei endopeptidase FOODPRO PAL Genencor/DuPont Acid fungal Aspergillus niger endopeptidase Metallo neutral Bacillus FOODPRO PNL Genencor/DuPont endopeptidase amyloliquefaciens FOODPRO Alkaline Alkaline Serine Bacillus Endopeptidase Protease Genencor/DuPont licheniformis FOODPRO PBR Genencor/DuPont Bromelain Ananas comosus Thermostable FOODPRO PHT Genencor/DuPont bacterial neutralGeobacillus sp.
endopeptidase FOODPRO 30L Genencor/DuPont Alkaline Serine Endopeptidase FOODPRO 51FP Genencor/DuPont Endo-/Exopeptidase FOODPRO PXT Genencor/DuPont subtilisin B. lentus ESPERASE 8.0L Novozymes protease Bacillus sp.
EVERLASE 16.0 subtilisin Bacillus sp.
ALCALASE 2.4 Novozymes subtilisin Bacillus sp.
NEUTRASE 0.8L Novozymes protease B.
amyloliquefaciens Allzyme FD Alltech Serine protease* Aspergillus niger Serratia proteamacula ns Arazyme One-Q Insect Biotech Co. metalloprotease HY-3 SAVINASE Novozymes subtilisin Bacillus sp.
Nocardiopsis prasina gene expressed in Alkaline serine Bacillus RONOZYME ProAct DSM/Novozymes protease licheniformis VALKERASE /CIBENZA Bacillus IND900 Novus Keratinase licheniformis
Commercial product ., Company Protease type Protease source ..
ALPHALASE AFP Genencor/DuPont Acid fungal Trichoderma reesei endopeptidase FOODPRO PAL Genencor/DuPont Acid fungal Aspergillus niger endopeptidase Metallo neutral Bacillus FOODPRO PNL Genencor/DuPont endopeptidase amyloliquefaciens FOODPRO Alkaline Alkaline Serine Bacillus Endopeptidase Protease Genencor/DuPont licheniformis FOODPRO PBR Genencor/DuPont Bromelain Ananas comosus Thermostable FOODPRO PHT Genencor/DuPont bacterial neutralGeobacillus sp.
endopeptidase FOODPRO 30L Genencor/DuPont Alkaline Serine Endopeptidase FOODPRO 51FP Genencor/DuPont Endo-/Exopeptidase FOODPRO PXT Genencor/DuPont subtilisin B. lentus ESPERASE 8.0L Novozymes protease Bacillus sp.
EVERLASE 16.0 subtilisin Bacillus sp.
ALCALASE 2.4 Novozymes subtilisin Bacillus sp.
NEUTRASE 0.8L Novozymes protease B.
amyloliquefaciens Allzyme FD Alltech Serine protease* Aspergillus niger Serratia proteamacula ns Arazyme One-Q Insect Biotech Co. metalloprotease HY-3 SAVINASE Novozymes subtilisin Bacillus sp.
Nocardiopsis prasina gene expressed in Alkaline serine Bacillus RONOZYME ProAct DSM/Novozymes protease licheniformis VALKERASE /CIBENZA Bacillus IND900 Novus Keratinase licheniformis
[0072] Additionally, or in the alternative endo- and/or exo-peptidase enzymes may be comprised in one or more of the following commercially available products: KANNASETm , NOVOCARNETM Tender' and Novozym 37020, NOVO-PROTM D (all available from Novozymes); BioSorb-ACDP (Noor Creations, India); ANGEL Acid Protease (Angel Yeast Co, Ltd., China) or COROLASE LAP (from AB Enzymes).
[0073] In some embodiments, the invention also provides a feed and/or food additive composition comprising at least one of the aminopeptidases described herein.
[0074] In another embodiment there is provided a composition and/or food additive and/or feed additive composition comprising a hydrolysate of the invention. Suitably, such a food and/or feed additive composition may further comprise an aminopeptidase (optionally in combination with an endoprotease).
[0075] Materials may be added to an enzyme-containing liquid to improve the properties of the liquid composition. Non-limiting examples of such additives include: salts (e.g., alkali salts, earth metal salts, additional chloride salts, sulfate salts, nitrate salts, carbonate salts, where exemplary counter ions are calcium, potassium, and sodium), inorganic minerals or clays (e.g., zeolites, kaolin, bentonite, talc's and/or silicates), carbohydrates (e.g., sucrose and/or starch), coloring pigments (e.g., titanium dioxide), biocides (e.g., RODALON , PROXEL
), dispersants, anti-foaming agents, reducing agents, acid agents, alkaline agents, enzyme stabilizers (e.g. polyol such as glycerol, propylene glycol, sorbitol, inorganic salts, sugars, sugar or a sugar alcohol, lactic acid, boric acid, or a boric acid derivative and combinations thereof), enzyme inhibitors, preservative (e.g. methyl paraben, propyl paraben, benzoate, sorbate or other food approved preservatives) and combinations thereof. Excipients which may be used in the preparation/composition include maltose, sucrose, glucose including glucose syrup or dried glucose syrup, pre-cooked starch, gelatinised starch, L-lactic, ascorbyl palmitate, tocopherols, lecithins, citric acid, citrates, phosphoric, phosphates, sodium alginate, carrageenan, locust bean gum, guar gum, xanthan gum, pectins, sodium carboxymethylcellulose, mono- and diglycerides, citric acid esters of mono- and diglycerides, sucrose esters, carbon dioxide, argon, helium, nitrogen, nitrous oxide, oxygen, hydrogen, and starch sodium octenylsuccinate.
As demonstrated in Example 7 herein, an aminopeptidase according to the present invention may retain its enzymatic activity in a composition comprising sodium chloride.
Forms
), dispersants, anti-foaming agents, reducing agents, acid agents, alkaline agents, enzyme stabilizers (e.g. polyol such as glycerol, propylene glycol, sorbitol, inorganic salts, sugars, sugar or a sugar alcohol, lactic acid, boric acid, or a boric acid derivative and combinations thereof), enzyme inhibitors, preservative (e.g. methyl paraben, propyl paraben, benzoate, sorbate or other food approved preservatives) and combinations thereof. Excipients which may be used in the preparation/composition include maltose, sucrose, glucose including glucose syrup or dried glucose syrup, pre-cooked starch, gelatinised starch, L-lactic, ascorbyl palmitate, tocopherols, lecithins, citric acid, citrates, phosphoric, phosphates, sodium alginate, carrageenan, locust bean gum, guar gum, xanthan gum, pectins, sodium carboxymethylcellulose, mono- and diglycerides, citric acid esters of mono- and diglycerides, sucrose esters, carbon dioxide, argon, helium, nitrogen, nitrous oxide, oxygen, hydrogen, and starch sodium octenylsuccinate.
As demonstrated in Example 7 herein, an aminopeptidase according to the present invention may retain its enzymatic activity in a composition comprising sodium chloride.
Forms
[0076] The product and/or the composition of the present invention may be used in any suitable form ¨ whether when alone or when present in a composition. Likewise, aminopeptidase of the present invention when combined with, e.g. exo- and/or endo- proteases may be used in any suitable form for use in the food industry as a food processing aid or foodstuff additive (i.e.
ingredients ¨ such as food ingredients, functional food ingredients or pharmaceutical ingredients).
ingredients ¨ such as food ingredients, functional food ingredients or pharmaceutical ingredients).
[0077] Suitable examples of forms include one or more of: tablets, pills, capsules, ovules, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.
[0078] By way of example, if the product and/or the composition are used in a tablet form ¨ such as for use as a functional ingredient ¨ the tablets may also contain one or more of: excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine; disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates;
granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia; lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia; lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
[0079] Examples of nutritionally acceptable carriers for use in preparing the forms include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.
[0080] Preferred excipients for the forms include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.
[0081] For aqueous suspensions and/or elixirs, aminopeptidase and/or the composition of the present invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerine, and combinations thereof.
[0082] The forms may also include gelatin capsules; fibre capsules, fibre tablets etc.
Methods
Methods
[0083] In some embodiments, the polypeptides of the present invention may be used in the production of protein hydrolysates, e.g., for enhancing the degree of hydrolysis, general debittering of protein hydrolysates and enhancing flavor development, production of glutamate and/or other uses like FAN generation during malting or brewing.
[0084] The present invention further relates to methods for using a polypeptide of the present invention in combination with a protease (e.g., an endopeptidase) to produce a high degree of hydrolysis of a protein-rich material. The method comprises treating of a proteinaceous substrate with the polypeptide and an endopeptidase. The substrate may be treated with the enzymes concurrently or consecutively.
[0085] A polypeptide of the present invention is added to the proteinaceous substrate in an effective amount conventionally employed in protein hydrolysis processes. In some embodiments, a polypeptide of the present invention is added to the proteinaceous substrate in the range of from about 0.1 to about 100,000 aminopeptidase units per 100 g of protein, or in the range of from about 1 to about 10,000 aminopeptidase units per 100 g of protein. As defined herein, one aminopeptidase unit (APU) is the amount of enzyme needed to release 1 micromole of p-nitroanilide per minute from Ala-p-nitroanilide (Sigma Chemical Co., St.
Louis MO) under the specified conditions. In the aminopeptidase assay the hydrolysis of the peptide substrate H-Ala¨nitroanilide is measured by the release of p¨nitroanilid (pNA). The absorbance of pNA is determined at a wavelength of 405 nm using an ELISA reader. The reaction is run with 18011.1 20 mM CPB buffer, 1511.1 diluted enzyme and 2011.1 substrate at 30 C. The CPB-Buffer is made up of 20mM Citric acid, 20mM Phosphate, 20mM Boric acid and adjusted to pH 9Ø
The substrate is 20 mg H-Ala-pNA from BACHEM (L-1070) in 1 ml DMSO (Dimethyl Sulphoxide from SIGMA (catalog # D2650).
Louis MO) under the specified conditions. In the aminopeptidase assay the hydrolysis of the peptide substrate H-Ala¨nitroanilide is measured by the release of p¨nitroanilid (pNA). The absorbance of pNA is determined at a wavelength of 405 nm using an ELISA reader. The reaction is run with 18011.1 20 mM CPB buffer, 1511.1 diluted enzyme and 2011.1 substrate at 30 C. The CPB-Buffer is made up of 20mM Citric acid, 20mM Phosphate, 20mM Boric acid and adjusted to pH 9Ø
The substrate is 20 mg H-Ala-pNA from BACHEM (L-1070) in 1 ml DMSO (Dimethyl Sulphoxide from SIGMA (catalog # D2650).
[0086] The endopeptidase may be obtained from a strain of Bacillus, preferably Bacillus licheniformis or Bacillus subtilis, a strain of Staphylococcus, preferably Staphylococcus aureus, a strain of Streptomyces, preferably Streptomyces thermovularis or Streptomyces griseus, a strain of Actinomyces species, a strain of Aspergillus, preferably Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae, or a strain of Trichoderma, preferably Trichoderma reesei, or Fusarium, preferably Fusarium venenatum. In some embodiments, the endopeptidase is selected from the group consisting of ALPHALASE AFP, FOODPRO PAL, FOODPRO PNL, FOODPRO Alkaline Protease, FOODPRO PXT, FOODPRO PBR, FOODPRO PHT, FOODPRO 30L, and
[0087] The endopeptidase is added to the proteinaceous substrate in an effective amount conventionally employed in protein hydrolysis processes, preferably in the range of from about 0.05 to about 15 AU/100 g of protein, and more preferably from about 0.1 to about 8 AU/100 g of protein. One AU (Anson Unit) is defined as the amount of enzyme which under standard conditions (i.e., 25 C, pH 7.5 and 10 min. reaction time) digests hemoglobin at an initial rate such that there is liberated per minute an amount of TCA soluble product which gives the same color with phenol reagent as one milliequivalent of tyrosine. In some embodiments, the endopeptidase may be dosed in an amount of about 10 to about 3000 mg of enzyme per kg of protein substrate, e.g. 0.01 to 3 g of enzyme per metric ton (MT) of protein substrate.
[0088] The enzymatic treatment, i.e., the incubation of the substrate with the enzyme preparations, may take place at any convenient temperature at which the enzyme preparation does not become inactivated, preferably in the range of from about 20 C to about 70 C. In accordance with established practice, the enzyme preparations may be suitably inactivated by increasing the temperature of the incubation mixture to a temperature where the enzymes become inactivated, e.g., to above about 70 C, or similarly by decreasing the pH of the incubation mixture to a point where the enzymes become inactivated, e.g., below about 4Ø
[0089] Furthermore, the methods of the present invention result in enhancement of the degree of hydrolysis of a proteinaceous substrate. As used herein, the degree of hydrolysis (DH) is the percentage of the total number of amino bonds in a protein that has been hydrolyzed by a proteolytic enzyme. In one aspect, an enzyme according to the present invention may facilitate an increase or enhancement of at least about 5, 7, 10, 12, 15, 17, 20, 22, 25, 27, 30, 35, 40, 45 or 50% of the DH of a proteinaceous substrate. In one example the increase may be relative to a type 1 aminopeptidase, although any suitable comparison may be made. In a preferred embodiment, the protein hydrolysates have an increased content of Leu, Gly, Glu, Ser, Asp, Asn, Pro, Cys, Ala, and/or Gln, e.g., at least 1.1 times greater.
[0090] In some embodiments, the protein hydrolysates have an increased content of Glu. In some embodiments, the protein hydrolysates have an increased content of Leu.
In some embodiments, the protein hydrolysates have an increased content of Gly. In some embodiments, the protein hydrolysates have an increased content of Ser. In some embodiments, the protein hydrolysates have an increased content of Asp. In some embodiments, the protein hydrolysates have an increased content of Asn. In some embodiments, the protein hydrolysates have an increased content of Pro. In some embodiments, the protein hydrolysates have an increased content of Cys. In some embodiments, the protein hydrolysates have an increased content of Ala.
In another more preferred embodiment, the protein hydrolysates have an increased content of Gln.
In some embodiments, the protein hydrolysates have an increased content of Gly. In some embodiments, the protein hydrolysates have an increased content of Ser. In some embodiments, the protein hydrolysates have an increased content of Asp. In some embodiments, the protein hydrolysates have an increased content of Asn. In some embodiments, the protein hydrolysates have an increased content of Pro. In some embodiments, the protein hydrolysates have an increased content of Cys. In some embodiments, the protein hydrolysates have an increased content of Ala.
In another more preferred embodiment, the protein hydrolysates have an increased content of Gln.
[0091] Aminopeptidase enzymes according to the present invention may exhibit decreased inhibition from the product of the enzymatic reaction. As demonstrated in Example 8 herein, two PepN 2 enzymes according to the present invention exhibited less inhibition by product than the enzyme leucine aminopeptidase 2 from Aspergillus oryzae RIB40 (NCBI
Reference Sequence: XP 001819545.1), which is set out below as SEQ ID NO:17:
1 mrsllwasll sgvlagralv spdefpediq ledllegsgq ledfayaype rnrvfggkah 61 ddtvnylyee lkktgyydvy kqpqvhlwsn adqtlkvgde eieaktmtys psvevtadva 121 vvknlgcsea dypsdvegkv alikrgecpf gdksvlaaka kaaasivynn vagsmagtlg 181 aaqsdkgpys aivgisledg qkliklaeag svsvdlwvds kqenrttynv vaqtkggdpn 241 nvvalgghtd sveagpgind dgsgiisnlv iakaltqysv knavrflfwt aeefgllgsn 301 yyvshlnate lnkirlylnf dmiaspnyal miydgdgsaf nqsgpagsaq ieklfedyyd 361 sidlphiptq fdgrsdyeaf ilngipsggl ftgaegimse enasrwggqa gvaydanyha 421 agdnmtnlnh eaflinskat afavatyand lssipkrntt sslhrrartm rpfgkrapkt 481 hahvsgsgcw hsgvea
Reference Sequence: XP 001819545.1), which is set out below as SEQ ID NO:17:
1 mrsllwasll sgvlagralv spdefpediq ledllegsgq ledfayaype rnrvfggkah 61 ddtvnylyee lkktgyydvy kqpqvhlwsn adqtlkvgde eieaktmtys psvevtadva 121 vvknlgcsea dypsdvegkv alikrgecpf gdksvlaaka kaaasivynn vagsmagtlg 181 aaqsdkgpys aivgisledg qkliklaeag svsvdlwvds kqenrttynv vaqtkggdpn 241 nvvalgghtd sveagpgind dgsgiisnlv iakaltqysv knavrflfwt aeefgllgsn 301 yyvshlnate lnkirlylnf dmiaspnyal miydgdgsaf nqsgpagsaq ieklfedyyd 361 sidlphiptq fdgrsdyeaf ilngipsggl ftgaegimse enasrwggqa gvaydanyha 421 agdnmtnlnh eaflinskat afavatyand lssipkrntt sslhrrartm rpfgkrapkt 481 hahvsgsgcw hsgvea
[0092] As such, in one embodiment the invention provides an isolated polypeptide that has aminopeptidase activity that has decreased product inhibition compared to the aminopeptidase of SEQ ID NO:17 (which is also referred to as TRI063 herein ¨
see e.g. the Examples). In particular, an isolated polypeptide according to the present invention that has aminopeptidase activity may have a product inhibition constant (Ki) value of greater than 2, 3, 4 or 5 mM. In a preferred embodiment the isolated polypeptide that has aminopeptidase activity is an aminopeptidase type 2 enzyme. In a further preferred embodiment the aminopeptidase type 2 enzyme is as set out in any of SEQ ID NOs:1-8 as defined herein, particularly SEQ ID NO:1 or 5.
see e.g. the Examples). In particular, an isolated polypeptide according to the present invention that has aminopeptidase activity may have a product inhibition constant (Ki) value of greater than 2, 3, 4 or 5 mM. In a preferred embodiment the isolated polypeptide that has aminopeptidase activity is an aminopeptidase type 2 enzyme. In a further preferred embodiment the aminopeptidase type 2 enzyme is as set out in any of SEQ ID NOs:1-8 as defined herein, particularly SEQ ID NO:1 or 5.
[0093] Aminopeptidase enzymes according to the present invention may be capable of hydrolyzing a polypeptide with a proline residue in position 2, as numbered from the N-terminus.
In particular, aminopeptidase enzymes according to the present invention may be capable of hydrolyzing a polypeptide with a proline residue in position 2, as numbered from the N-terminus, to over half the starting concentration within 2 hours of incubation. The degree of hydrolysis can be measured by any suitable protocol known in the art, for example, the method presented in Example 10. As demonstrated in Example 10, two PepN 2 enzymes according to the present invention (TRI032 and TRI035) are able to hydrolyze the peptide TPAAAR over time to less than half the concentration within 2 hr incubation, whereas TRI063 (A. oryzae) and COROLASE LAP show no hydrolysis of TPAAAR within 12 h.
In particular, aminopeptidase enzymes according to the present invention may be capable of hydrolyzing a polypeptide with a proline residue in position 2, as numbered from the N-terminus, to over half the starting concentration within 2 hours of incubation. The degree of hydrolysis can be measured by any suitable protocol known in the art, for example, the method presented in Example 10. As demonstrated in Example 10, two PepN 2 enzymes according to the present invention (TRI032 and TRI035) are able to hydrolyze the peptide TPAAAR over time to less than half the concentration within 2 hr incubation, whereas TRI063 (A. oryzae) and COROLASE LAP show no hydrolysis of TPAAAR within 12 h.
[0094] The present invention also relates to methods for obtaining a protein hydrolysate enriched in free glutamic acid and/or peptide bound glutamic acid residues, which method comprises: subjecting the substrate to the action of a polypeptide having aminopeptidase activity.
The present invention also relates to methods for obtaining a protein hydrolysate enriched in free glutamine or glutamic acid and/or oligopeptide bound glutamine or glutamic acid residues, which method comprises: subjecting the substrate to the action of a polypeptide having aminopeptidase activity.
The present invention also relates to methods for obtaining a protein hydrolysate enriched in free glutamine or glutamic acid and/or oligopeptide bound glutamine or glutamic acid residues, which method comprises: subjecting the substrate to the action of a polypeptide having aminopeptidase activity.
[0095] The present invention also relates to methods for debittering a protein hydrolysate, which method comprises: subjecting the substrate to the action of a polypeptide having aminopeptidase activity.
[0096] In some embodiments, the methods further comprise subjecting the substrate to a deamidation process. The deamidation process may be performed simultaneously, prior or subsequently to the subjecting the substrate to the action of a polypeptide having aminopeptidase activity.
[0097] In some embodiments, the methods of the present invention produce protein hydrolysates with enhanced flavor because glutamic acid (Glu), whether free or oligopeptide bound, plays an important role in the flavor and palatability of protein hydrolysates. In some embodiments, the method also produces protein hydrolysates having improved functionality, in particular, improved solubility, improved emulsifying properties, increased degree of hydrolysis, and improved foaming properties.
[0098] The conversion of amides (glutamine or asparagine) into charged acids (glutamic acid or aspartic acid) via the liberation of ammonia is known as deamidation.
Deamidation may take place as a non-enzymatic or as an enzymatic deamidation process.
Deamidation may take place as a non-enzymatic or as an enzymatic deamidation process.
[0099] In some embodiments, the deamidation is carried out as an enzymatic deamidation process, e.g., by subjecting the substrate to a glutaminase, transglutaminase and/or peptidoglutaminase.
[00100] In some embodiments the glutaminase is GLUTAMINASE SD-ClOOSTm (Amano, Japan).
[00101] In some embodiments, the glutaminase may be dosed in an amount of about 1 mg to 20 mg per g of substrate protein. In some embodiments, the glutaminase may be dosed in an amount of about 5 mg to 15 mg per g of substrate protein. In some embodiments, the glutaminase may be dosed in an amount of about 10 mg per g of substrate protein.
[00102] The present invention also relates to methods for the production of glutamate, which method comprises: subjecting a protein substrate to the action of a polypeptide having aminopeptidase activity.
[00103] The transglutaminase may be of any convenient source including mammals, see e.g., JP 1050382 and JP 5023182, including activated Factor XIII, see e.g., WO
93/15234; those derived from fish, see e.g., EP 555,649; and those obtained from microorganisms, see e.g., EP
379,606, WO 96/06931 and WO 96/22366. In some embodiments, the transglutaminase is obtained from an Oomycete, including a strain of Phytophthora, preferably Phytophthora cactorum, or a strain of Pythium, preferably Pythium irregulare, Pythium sp., Pythium intermedium, Pythium it/mum, or Pythium periilum (or Pythium periplocum). In some embodiments, the transglutaminase is of bacterial origin and is obtained from a strain of Bacillus, preferably Bacillus subtilis, a strain of Streptoverticillium, preferably Streptoverticillium mobaraensis, Streptoverticillium griseocarneum, or Streptoverticillium cinnamoneum, and a strain of Streptomyces, preferably Streptomyces lydicus.
93/15234; those derived from fish, see e.g., EP 555,649; and those obtained from microorganisms, see e.g., EP
379,606, WO 96/06931 and WO 96/22366. In some embodiments, the transglutaminase is obtained from an Oomycete, including a strain of Phytophthora, preferably Phytophthora cactorum, or a strain of Pythium, preferably Pythium irregulare, Pythium sp., Pythium intermedium, Pythium it/mum, or Pythium periilum (or Pythium periplocum). In some embodiments, the transglutaminase is of bacterial origin and is obtained from a strain of Bacillus, preferably Bacillus subtilis, a strain of Streptoverticillium, preferably Streptoverticillium mobaraensis, Streptoverticillium griseocarneum, or Streptoverticillium cinnamoneum, and a strain of Streptomyces, preferably Streptomyces lydicus.
[00104] The peptidoglutaminase may be a peptidoglutaminase I (peptidyl-glutaminase; EC
3.5.1.43), or a peptidoglutaminase II (protein-glutamine glutaminase; EC
3.5.1.44), or any mixture thereof. The peptidoglutaminase may be obtained from a strain of Aspergillus, preferably Aspergillus japonicus, a strain of Bacillus, preferably Bacillus circulans, a strain of Cryptococcus, preferably Cryptococcus albidus, or a strain of Debaryomyces, preferably Debaryomyces kloecheri.
3.5.1.43), or a peptidoglutaminase II (protein-glutamine glutaminase; EC
3.5.1.44), or any mixture thereof. The peptidoglutaminase may be obtained from a strain of Aspergillus, preferably Aspergillus japonicus, a strain of Bacillus, preferably Bacillus circulans, a strain of Cryptococcus, preferably Cryptococcus albidus, or a strain of Debaryomyces, preferably Debaryomyces kloecheri.
[00105] The transglutaminase is added to the proteinaceous substrate in an effective amount conventionally employed in deamidation processes, preferably in the range of from about 0.01 to about 5% (w/w), and more preferably in the range of from about 0.1 to about 1 %
(w/w) of enzyme preparation relating to the amount of substrate.
(w/w) of enzyme preparation relating to the amount of substrate.
[00106] The peptidoglutaminase is added to the proteinaceous substrate in an effective amount conventionally employed in deamidation processes, preferably in the range of from about 0.01 to about 100,000 PGase Units per 100 g of substrate, and more preferably in the range of from about 0.1 to about 10,000 PGase Units per 100 g of substrate.
[00107] The peptidoglutaminase activity may be determined according to the procedure of Cedrangoro et at. (1965, Enzymologia Vol. 29 page143). According to this procedure, 0.5 ml of an enzyme sample, adjusted to pH 6.5 with 1 N NaOH, is charged into a small vessel. Then 1 ml of a borate pH 10.8 buffer solution is added to the vessel. The discharged ammonia is absorbed by 5 N sulphuric acid, and by use of Nessler's reagent the mixture is allowed to form color which is measured at 420 nm. One PGase unit is the amount of enzyme capable of producing 1 micromole of ammonia per minute under these conditions.
[00108] The present invention also relates to methods for the production of free amino nitrogen (FAN) during malting and/or brewing, which method comprises:
subjecting a substrate during a malting and/or brewing process to the action of a polypeptide having aminopeptidase activity.
subjecting a substrate during a malting and/or brewing process to the action of a polypeptide having aminopeptidase activity.
[00109] In some embodiments of the methods of the present invention, a protein substrate is subjected to a polypeptide of the present invention. A polypeptide of the present invention is added to the proteinaceous substrate in an effective amount conventionally employed in protein hydrolysis processes, preferably in the range of from about 0.001 to about 0.5 AU/100 g of substrate, more preferably in the range of from about 0.01 to about 0.1 AU/100 g of substrate.
[00110] In another embodiment, the methods of the present invention for producing a hydrolysate enriched in free glutamic acid and/or peptide bound glutamic acid residues further comprise: subjecting the substrate to one or more unspecific acting endo-and/or exo-peptidase enzymes. This step may take place simultaneously, or may follow the step of subjecting a protein substrate with a polypeptide of the present invention.
[00111] In a preferred embodiment, the unspecific acting endo- and/or exo-peptidase enzyme is obtained from a strain of Aspergillus, or a strain of Bacillus.
[00112] The unspecific acting endo- and/or exo-peptidase enzyme is added to the substrate in an effective amount conventionally employed in protein hydrolysis processes, preferably in the range of from about.
[00113] In some embodiments, the endo- and/or exo-peptidase may be dosed in an amount of about 50 to about 3000 mg of enzyme per kg of protein substrate, e.g. 0.05 to 3 g of enzyme per metric ton (MT) of protein substrate.
[00114] Suitably, the endo- and/or exo-peptidase may be dosed in an amount of less than about 4.0 g of enzyme per MT of protein substrate.
[00115] In another embodiment, the endo- and/or exo-peptidase may be dosed at between about 0.5 g and about 5.0 g of enzyme per MT of protein substrate. Suitably the endo- and/or exo-peptidase may be dosed at between about 0.5 g and about 3.0 g of enzyme per MT of protein substrate. More suitably, the endoprotease may be dosed at about 1.0 g to about 2.0 g of enzyme per MT of protein substrate.
[00116] In some embodiments, a polypeptide of the present invention may be dosed in an amount of between about 0.5 mg to about 2 g of enzyme per kg of protein substrate and/or food and/or feed additive composition. Suitably a polypeptide of the present invention may be dosed in an amount of between about 1 mg to about 2 g of enzyme per kg of protein substrate and/or food and/or feed additive composition. More suitably in an amount of between about 5 mg to about 1.5 g of enzyme per kg of protein substrate and/or food and/or feed additive composition.
[00117] In the preparation of a hydrolysate a polypeptide of the present invention may be dosed in an amount of between about 0.5 mg to about 2 g of enzyme per kg of protein substrate.
Suitably a polypeptide of the present invention may be dosed in an amount of between about 1 mg to about 2 g of enzyme per kg of protein substrate. More suitably in an amount of between about 5 mg to about 1.5 g of enzyme per kg of protein substrate.
Suitably a polypeptide of the present invention may be dosed in an amount of between about 1 mg to about 2 g of enzyme per kg of protein substrate. More suitably in an amount of between about 5 mg to about 1.5 g of enzyme per kg of protein substrate.
[00118] In one embodiment, a polypeptide of the present invention may be dosed in an amount of between about 5 mg to about 500 mg of enzyme per kg of protein substrate. Suitably a polypeptide of the present invention may be dosed in an amount of between about 50 mg to about 500 mg of enzyme per kg of protein substrate. Suitably a polypeptide of the present invention may be dosed in an amount of between about 100 mg to about 450 mg of enzyme per kg of protein substrate.
[00119] Each enzymatic treatment may take place at any temperature at which the enzyme preparation does not become inactivated, preferably in the range of from about 20 C to about 70 C. The enzyme preparation may then be inactivated by increasing the temperature, e.g., to above about 70 C, or by decreasing the pH, e.g., below about 4Ø
[00120] The proteinaceous substrate used in the methods of the present invention may consist of intact proteins, prehydrolyzed proteins (i.e., peptides), or a mixture thereof. The proteinaceous substrate may be of vegetable or animal origin. In some embodiments, the proteinaceous substrate is of vegetable origin, e.g., soy protein, grain protein, e.g., wheat gluten, corn gluten, barley, rye, oat, rice, zein, lupine, cotton seed protein, rape seed protein, peanut, alfalfa protein, pea protein, fabaceous bean protein, sesame seed protein, or sunflower. A
proteinaceous substrate of animal origin may be whey protein, casein, meat proteins, fish protein, red blood cells, egg white, gelatin, lactoalbumin, hair proteins or feather proteins.
proteinaceous substrate of animal origin may be whey protein, casein, meat proteins, fish protein, red blood cells, egg white, gelatin, lactoalbumin, hair proteins or feather proteins.
[00121] The present invention also relates to protein hydrolysates produced by these methods.
Preparation of the nucleotide sequence
Preparation of the nucleotide sequence
[00122] A nucleotide sequence encoding either a protein which has the specific properties as defined herein or a protein which is suitable for modification may be identified and/or isolated and/or purified from any cell or organism producing said protein. Various methods are well known within the art for the identification and/or isolation and/or purification of nucleotide sequences. By way of example, PCR amplification techniques to prepare more of a sequence may be used once a suitable sequence has been identified and/or isolated and/or purified.
[00123] By way of further example, a genomic DNA and/or cDNA library may be constructed using chromosomal DNA or messenger RNA from the organism producing the enzyme. If the amino acid sequence of the enzyme is known, labelled oligonucleotide probes may be synthesized and used to identify enzyme-encoding clones from the genomic library prepared from the organism. Alternatively, a labelled oligonucleotide probe containing sequences homologous to another known enzyme gene could be used to identify enzyme-encoding clones. In the latter case, hybridization and washing conditions of lower stringency are used.
[00124] Alternatively, enzyme-encoding clones could be identified by inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming enzyme-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar plates containing a substrate for enzyme (i.e., maltose), thereby allowing clones expressing the enzyme to be identified.
[00125] In a yet further alternative, the nucleotide sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoramidite method described by Beucage S.L. et at., (1981) Tetrahedron Letters 22, p 1859-1869, or the method described by Matthes et at., (1984) EMBO J. 3, p 801-805. In the phosphoramidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
[00126] The nucleotide sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques.
Each ligated fragment corresponds to various parts of the entire nucleotide sequence. The DNA
sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or in Saiki R K et at., (Science (1988) 239, pp 487-491).
Amino acid sequences
Each ligated fragment corresponds to various parts of the entire nucleotide sequence. The DNA
sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or in Saiki R K et at., (Science (1988) 239, pp 487-491).
Amino acid sequences
[00127] The scope of the present invention also encompasses amino acid sequences of enzymes having the specific properties as defined herein.
[00128] As used herein, the term "amino acid sequence" is synonymous with the term c`polypeptide" and/or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "enzyme".
[00129] The amino acid sequence may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA
techniques.
techniques.
[00130] The protein encompassed in the present invention may be used in conjunction with other proteins, particularly proline endoprotease, tripeptidyl exopeptidases, and other forms of endo or exoproteases. Thus the present invention also covers a combination of proteins wherein the combination comprises the aminopeptidase of the present invention and another enzyme, which may be another aminopeptidase according to the present invention. This aspect is discussed in a later section.
[00131] Preferably the amino acid sequence when relating to and when encompassed by the per se scope of the present invention is not a native enzyme. In this regard, the term "native enzyme" means an entire enzyme that is in its native environment and when it has been expressed by its native nucleotide sequence.
Sequence identity or sequence homology
Sequence identity or sequence homology
[00132] The present invention also encompasses the use of sequences having a degree of sequence identity or sequence homology with amino acid sequence(s) of a polypeptide having the specific properties defined herein or of any nucleotide sequence encoding such a polypeptide (hereinafter referred to as a "homologous sequence(s)"). Here, the term "homologue" means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences. Here, the term "homology" can be equated with "identity".
[00133] The homologous amino acid sequence and/or nucleotide sequence should provide and/or encode a polypeptide which retains the functional activity and/or enhances the activity of the aminopeptidase.
[00134] In the present context, a homologous sequence is taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to the subject sequence. Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
[00135] In the present context, a homologous sequence is taken to include a nucleotide sequence which may be at least 75, 80, 85 or 90% identical, preferably at least 95 or 98%
identical to a nucleotide sequence encoding a polypeptide of the present invention (the subject sequence). Typically, the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
identical to a nucleotide sequence encoding a polypeptide of the present invention (the subject sequence). Typically, the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
[00136] Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percentage homology between two or more sequences.
[00137] Percentage homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
[00138] Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in percentage homology when a global alignment is performed.
Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalizing unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximize local homology.
Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalizing unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximize local homology.
[00139] However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system.
High gap penalties will of course produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons.
High gap penalties will of course produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons.
[00140] Calculation of maximum percentage homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A
suitable computer program for carrying out such an alignment is the Vector NTI (Invitrogen Corp.). Examples of software that can perform sequence comparisons include, but are not limited to, the BLAST
package (see Ausubel et at. 1999, Short Protocols in Molecular Biology, 4th Ed - Chapter 18), BLAST 2 (see FEMS Microbial Lett 1999 174(2): 247-50; FEMS Microbial Lett 1999 177(1):
187-8 and [email protected]), FASTA (Altschul et al, 19901 Mol. Biol.
403-410) and AlignX for example. At least BLAST, BLAST 2 and FASTA are available for offline and online searching (see Ausubel et at. 1999, supra, pages 7-58 to 7-60).
suitable computer program for carrying out such an alignment is the Vector NTI (Invitrogen Corp.). Examples of software that can perform sequence comparisons include, but are not limited to, the BLAST
package (see Ausubel et at. 1999, Short Protocols in Molecular Biology, 4th Ed - Chapter 18), BLAST 2 (see FEMS Microbial Lett 1999 174(2): 247-50; FEMS Microbial Lett 1999 177(1):
187-8 and [email protected]), FASTA (Altschul et al, 19901 Mol. Biol.
403-410) and AlignX for example. At least BLAST, BLAST 2 and FASTA are available for offline and online searching (see Ausubel et at. 1999, supra, pages 7-58 to 7-60).
[00141] Although the final percentage homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. Vector NTI programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the default values for the Vector NTI package.
[00142] Alternatively, percentage homologies may be calculated using the multiple alignment feature in Vector NTI (Invitrogen Corp.), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244).
[00143] Once the software has produced an optimal alignment, it is possible to calculate percentage homology, preferably percentage sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
[00144] Should Gap Penalties be used when determining sequence identity, then preferably the following parameters are used for pairwise alignment:
FOR BLAST
FOR CLUSTAL DNA PROTEIN
WORD SIZE 2 1 K triple GAP EXTENSION 6.66 0.1
FOR BLAST
FOR CLUSTAL DNA PROTEIN
WORD SIZE 2 1 K triple GAP EXTENSION 6.66 0.1
[00145] In one embodiment, CLUSTAL may be used with the gap penalty and gap extension set as defined above.
[00146] Suitably, the degree of identity with regard to a nucleotide sequence is determined over at least 20 contiguous nucleotides, preferably over at least 30 contiguous nucleotides, preferably over at least 40 contiguous nucleotides, preferably over at least 50 contiguous nucleotides, preferably over at least 60 contiguous nucleotides, preferably over at least 100 contiguous nucleotides.
[00147] Suitably, the degree of identity with regard to a nucleotide sequence may be determined over the whole sequence.
Variants/homologues/derivatives
Variants/homologues/derivatives
[00148] The present invention also encompasses the use of variants, homologues and derivatives of any amino acid sequence of a protein or of any nucleotide sequence encoding such a protein
[00149] Here, the term "homologue" means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences. Here, the term "homology"
can be equated with "identity".
can be equated with "identity".
[00150] In the present context, a homologous sequence is taken to include an amino acid sequence which may be at least 75, 80, 85 or 90% identical, preferably at least 95, 96, 97, 98 or 99% identical to the subject sequence. Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
[00151] In the present context, a homologous sequence is taken to include a nucleotide sequence which may be at least 75, 80, 85 or 90% identical, preferably at least 95, 96, 97, 98 or 99% identical to a nucleotide sequence encoding an enzyme of the present invention (the subject sequence). Typically, the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
[00152] Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percentage homology between two or more sequences.
Percentage homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment.
Typically, such ungapped alignments are performed only over a relatively short number of residues.
Percentage homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment.
Typically, such ungapped alignments are performed only over a relatively short number of residues.
[00153] Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in percentage homology when a global alignment is performed.
Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalizing unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximize local homology.
Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalizing unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximize local homology.
[00154] However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system.
High gap penalties will of course produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.
High gap penalties will of course produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.
[00155] Calculation of maximum percentage homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A
suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux et at 1984 Nuc. Acids Research 12 p387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et at., 1999, supra), FASTA (Altschul et at., 19901 Mot. Biol. 403-410) and the GENEWORKS
suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et at., 1999, supra). However, for some applications, it is preferred to use the GCG
Bestfit program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence.
suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux et at 1984 Nuc. Acids Research 12 p387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et at., 1999, supra), FASTA (Altschul et at., 19901 Mot. Biol. 403-410) and the GENEWORKS
suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et at., 1999, supra). However, for some applications, it is preferred to use the GCG
Bestfit program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence.
[00156] Although the final percentage homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG
Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
[00157] Alternatively, percentage homologies may be calculated using the multiple alignment feature in DNASISTm (Hitachi Software), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), supra).
[00158] Once the software has produced an optimal alignment, it is possible to calculate percentage homology, preferably percentage sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
[00159] The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance.
Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
[00160] Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
ALIPHATIC Non-polar G A P
I L V
Polar ¨ uncharged CSTM
NQ
Polar ¨ charged D E
KR
AROMATIC HFWY
ALIPHATIC Non-polar G A P
I L V
Polar ¨ uncharged CSTM
NQ
Polar ¨ charged D E
KR
AROMATIC HFWY
[00161] The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as 0), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.
[00162] Replacements may also be made by unnatural amino acids include;
alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, B-alanine*, L-a-amino butyric acid*, L-y-amino butyric acid*, L-a-amino isobutyric acid*, L-c-amino caproic acid, 7-amino heptanoic acid*, L-methionine sulfone#*, L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline#, L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino)#, L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionic acid # and L-Phe (4-benzyl)*.
The notation * has been utilized for the purpose of the discussion above (relating to homologous or non-homologous substitution), to indicate the hydrophobic nature of the derivative whereas #
has been utilized to indicate the hydrophilic nature of the derivative, #*
indicates amphipathic characteristics.
alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, B-alanine*, L-a-amino butyric acid*, L-y-amino butyric acid*, L-a-amino isobutyric acid*, L-c-amino caproic acid, 7-amino heptanoic acid*, L-methionine sulfone#*, L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline#, L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino)#, L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionic acid # and L-Phe (4-benzyl)*.
The notation * has been utilized for the purpose of the discussion above (relating to homologous or non-homologous substitution), to indicate the hydrophobic nature of the derivative whereas #
has been utilized to indicate the hydrophilic nature of the derivative, #*
indicates amphipathic characteristics.
[00163] Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or B-alanine residues. A further form of variation, involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, "the peptoid form" is used to refer to variant amino acid residues wherein the a-carbon sub stituent group is on the residue's nitrogen atom rather than the a-carbon.
Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ et at., PNAS
(1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13 (4), 132-134.
Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ et at., PNAS
(1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13 (4), 132-134.
[00164] The nucleotide sequences for use in the present invention may include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art.
Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences of the present invention.
Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences of the present invention.
[00165] The present invention also encompasses the use of nucleotide sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar coding sequences in other organisms etc.
[00166] Polynucleotides which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA
libraries made from a range of individuals, for example individuals from different populations.
In addition, other homologues may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridizing to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention.
libraries made from a range of individuals, for example individuals from different populations.
In addition, other homologues may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridizing to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention.
[00167] Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention.
Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example, the GCG Wisconsin PileUp program is widely used.
Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example, the GCG Wisconsin PileUp program is widely used.
[00168] The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
[00169] Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterized sequences. This may be useful where for example silent codon sequence changes are required to optimize codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.
[00170] Polynucleotides (nucleotide sequences) of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.
[00171] Polynucleotides such as DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
[00172] In general, primers will be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time.
Techniques for accomplishing these using automated techniques are readily available in the art.
Techniques for accomplishing these using automated techniques are readily available in the art.
[00173] Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.
Hybridization
Hybridization
[00174] The present invention also encompasses sequences that are complementary to the nucleic acid sequences of the present invention or sequences that are capable of hybridizing either to the sequences of the present invention or to sequences that are complementary thereto.
[00175] The term "hybridization" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing"
as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies.
as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies.
[00176] The present invention also encompasses the use of nucleotide sequences that are capable of hybridizing to the sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof.
[00177] The term "variant" also encompasses sequences that are complementary to sequences that are capable of hybridizing to the nucleotide sequences presented herein.
[00178] Preferably, the term "variant" encompasses sequences that are complementary to sequences that are capable of hybridizing under stringent conditions (e.g. 50 C and 0.2xSSC
{1xSSC = 0.15 M NaC1, 0.015 M Na3citrate pH 7.0}) to the nucleotide sequences presented herein.
{1xSSC = 0.15 M NaC1, 0.015 M Na3citrate pH 7.0}) to the nucleotide sequences presented herein.
[00179] More preferably, the term "variant" encompasses sequences that are complementary to sequences that are capable of hybridizing under high stringent conditions (e.g.
65 C and 0.1xSSC {1xSSC = 0.15 M NaC1, 0.015 M Na3citrate pH 7.0}) to the nucleotide sequences presented herein.
65 C and 0.1xSSC {1xSSC = 0.15 M NaC1, 0.015 M Na3citrate pH 7.0}) to the nucleotide sequences presented herein.
[00180] The present invention also relates to nucleotide sequences that can hybridize to the nucleotide sequences of the present invention (including complementary sequences of those presented herein).
[00181] The present invention also relates to nucleotide sequences that are complementary to sequences that can hybridize to the nucleotide sequences of the present invention (including complementary sequences of those presented herein).
[00182] Also included within the scope of the present invention are polynucleotide sequences that are capable of hybridizing to the nucleotide sequences presented herein under conditions of intermediate to maximal stringency.
[00183] In a preferred aspect, the present invention covers nucleotide sequences that can hybridize to the nucleotide sequence of the present invention, or the complement thereof, under stringent conditions (e.g. 50 C and 0.2xSSC).
[00184] In a more preferred aspect, the present invention covers nucleotide sequences that can hybridize to the nucleotide sequence of the present invention, or the complement thereof, under high stringent conditions (e.g. 65 C and 0.1xSSC).
Molecular evolution
Molecular evolution
[00185] As a non-limiting example, it is possible to produce numerous site directed or random mutations into a nucleotide sequence, either in vivo or in vitro, and to subsequently screen for improved functionality of the encoded polypeptide by various means.
[00186] In addition, mutations or natural variants of a polynucleotide sequence can be recombined with either the wildtype or other mutations or natural variants to produce new variants. Such new variants can also be screened for improved functionality of the encoded polypeptide. The production of new preferred variants can be achieved by various methods well established in the art, for example the Error Threshold Mutagenesis (WO
92/18645), oligonucleotide mediated random mutagenesis (US 5,723, 323), DNA shuffling (US
5,605,793), exo-mediated gene assembly W000/58517. The application of these and similar random directed molecular evolution methods allows the identification and selection of variants of the enzymes of the present invention which have preferred characteristics without any prior knowledge of protein structure or function, and allows the production of non-predictable but beneficial mutations or variants. There are numerous examples of the application of molecular evolution in the art for the optimization or alteration of enzyme activity, such examples include, but are not limited to one or more of the following:
optimized expression and/or activity in a host cell or in vitro, increased enzymatic activity, altered substrate and/or product specificity, increased or decreased enzymatic or structural stability, altered enzymatic activity/specificity in preferred environmental conditions, e.g.
temperature, pH, substrate Site-directed mutagenesis
92/18645), oligonucleotide mediated random mutagenesis (US 5,723, 323), DNA shuffling (US
5,605,793), exo-mediated gene assembly W000/58517. The application of these and similar random directed molecular evolution methods allows the identification and selection of variants of the enzymes of the present invention which have preferred characteristics without any prior knowledge of protein structure or function, and allows the production of non-predictable but beneficial mutations or variants. There are numerous examples of the application of molecular evolution in the art for the optimization or alteration of enzyme activity, such examples include, but are not limited to one or more of the following:
optimized expression and/or activity in a host cell or in vitro, increased enzymatic activity, altered substrate and/or product specificity, increased or decreased enzymatic or structural stability, altered enzymatic activity/specificity in preferred environmental conditions, e.g.
temperature, pH, substrate Site-directed mutagenesis
[00187] Once a protein-encoding nucleotide sequence has been isolated, or a putative protein-encoding nucleotide sequence has been identified, it may be desirable to mutate the sequence in order to prepare a protein of the present invention.
[00188] Mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites.
[00189] A suitable method is disclosed in Morinaga et al., (Biotechnology (1984) 2, p646-649). Another method of introducing mutations into enzyme-encoding nucleotide sequences is described in Nelson and Long (Analytical Biochemistry (1989), 180, p 147-151).
Recombinant
Recombinant
[00190] In one aspect the sequence for use in the present invention is a recombinant sequence ¨ i.e. a sequence that has been prepared using recombinant DNA
techniques.
techniques.
[00191] These recombinant DNA techniques are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature, for example, J.
Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A
Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press.
Synthetic
Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A
Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press.
Synthetic
[00192] In one aspect the sequence for use in the present invention is a synthetic sequence ¨ i.e. a sequence that has been prepared by in vitro chemical or enzymatic synthesis. It includes, but is not limited to, sequences made with optimal codon usage for host organisms - such as the methylotrophic yeasts Pichia and Hansenula.
Expression Expression of enzymes
Expression Expression of enzymes
[00193] The nucleotide sequence for use in the present invention may be incorporated into a recombinant replicable vector. The vector may be used to replicate and express the nucleotide sequence, in protein/enzyme form, in and/or from a compatible host cell.
[00194] Expression may be controlled using control sequences e.g.
regulatory sequences.
regulatory sequences.
[00195] The protein produced by a host recombinant cell by expression of the nucleotide sequence may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. The coding sequences may be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane.
Expression vector
Expression vector
[00196] The term "expression vector" means a construct capable of in vivo or in vitro expression.
[00197] Preferably, the expression vector is incorporated into the genome of a suitable host organism. The term "incorporated" preferably covers stable incorporation into the genome.
[00198] The nucleotide sequence of the present invention may be present in a vector in which the nucleotide sequence is operably linked to regulatory sequences capable of providing for the expression of the nucleotide sequence by a suitable host organism.
[00199] The vectors for use in the present invention may be transformed into a suitable host cell as described below to provide for expression of a polypeptide of the present invention.
[00200] The choice of vector e.g. a plasmid, cosmid, or phage vector will often depend on the host cell into which it is to be introduced.
[00201] The vectors for use in the present invention may contain one or more selectable marker genes- such as a gene, which confers antibiotic resistance e.g.
ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Alternatively, the selection may be accomplished by co-transformation (as described in W091/17243).
ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Alternatively, the selection may be accomplished by co-transformation (as described in W091/17243).
[00202] Vectors may be used in vitro, for example for the production of RNA
or used to transfect, transform, transduce or infect a host cell.
or used to transfect, transform, transduce or infect a host cell.
[00203] Thus, in a further embodiment, the invention provides a method of making nucleotide sequences of the present invention by introducing a nucleotide sequence of the present invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
[00204] The vector may further comprise a nucleotide sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.
Regulatory sequences
Regulatory sequences
[00205] In some applications, the nucleotide sequence for use in the present invention is operably linked to a regulatory sequence which is capable of providing for the expression of the nucleotide sequence, such as by the chosen host cell. By way of example, the present invention covers a vector comprising the nucleotide sequence of the present invention operably linked to such a regulatory sequence, i.e. the vector is an expression vector.
[00206] The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A
regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
[00207] The term "regulatory sequences" includes promoters and enhancers and other expression regulation signals.
[00208] The term "promoter" is used in the normal sense of the art, e.g. an RNA
polymerase binding site.
polymerase binding site.
[00209] Enhanced expression of the nucleotide sequence encoding the enzyme of the present invention may also be achieved by the selection of heterologous regulatory regions, e.g.
promoter, secretion leader and terminator regions.
promoter, secretion leader and terminator regions.
[00210] Preferably, the nucleotide sequence according to the present invention is operably linked to at least a promoter.
[00211] Other promoters may even be used to direct expression of the polypeptide of the present invention.
[00212] Examples of suitable promoters for directing the transcription of the nucleotide sequence in a bacterial, fungal or yeast host are well known in the art.
[00213] The promoter can additionally include features to ensure or to increase expression in a suitable host. For example, the features can be conserved regions such as a Pribnow Box or a TATA box.
Constructs
Constructs
[00214] The term "construct" - which is synonymous with terms such as "conjugate", "cassette" and "hybrid" - includes a nucleotide sequence for use according to the present invention directly or indirectly attached to a promoter.
[00215] An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Shl-intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention. The same is true for the term "fused" in relation to the present invention which includes direct or indirect attachment. In some cases, the terms do not cover the natural combination of the nucleotide sequence coding for the protein ordinarily associated with the wild type gene promoter and when they are both in their natural environment.
[00216] The construct may even contain or express a marker, which allows for the selection of the genetic construct.
[00217] For some applications, preferably the construct of the present invention comprises at least the nucleotide sequence of the present invention operably linked to a promoter.
Host cells
Host cells
[00218] The term "host cell" - in relation to the present invention includes any cell that comprises either the nucleotide sequence or an expression vector as described above and which is used in the recombinant production of a protein having the specific properties as defined herein.
[00219] Thus, a further embodiment of the present invention provides host cells transformed or transfected with a nucleotide sequence that expresses the protein of the present invention. The cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells.
[00220] Examples of suitable bacterial host organisms are gram positive or gram negative bacterial species.
[00221] Depending on the nature of the nucleotide sequence encoding the polypeptide of the present invention, and/or the desirability for further processing of the expressed protein, eukaryotic hosts such as yeasts or other fungi may be preferred. In general, yeast cells are preferred over fungal cells because they are easier to manipulate. However, some proteins are either poorly secreted from the yeast cell, or in some cases are not processed properly (e.g.
hyperglycosylation in yeast). In these instances, a different fungal host organism should be selected.
hyperglycosylation in yeast). In these instances, a different fungal host organism should be selected.
[00222] The use of suitable host cells - such as yeast, fungal and plant host cells - may provide for post-translational modifications (e.g. myristoylation, glycosylation, truncation, lipidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.
[00223] The host cell may be an aminopeptidase deficient or aminopeptidase minus strain.
The present invention also relates to methods for producing a mutant cell of a parent cell, which comprises disrupting or deleting a nucleic acid sequence encoding the polypeptide or a control sequence thereof, which results in the mutant cell producing less of the polypeptide than the parent cell.
The present invention also relates to methods for producing a mutant cell of a parent cell, which comprises disrupting or deleting a nucleic acid sequence encoding the polypeptide or a control sequence thereof, which results in the mutant cell producing less of the polypeptide than the parent cell.
[00224] The construction of strains which have reduced aminopeptidase activity may be conveniently accomplished by modification or inactivation of a nucleic acid sequence necessary for expression of the polypeptide having aminopeptidase activity in the cell.
Organism
Organism
[00225] The term "organism" in relation to the present invention includes any organism that could comprise the nucleotide sequence coding for the polypeptide according to the present invention and/or products obtained therefrom, and/or wherein a promoter can allow expression of the nucleotide sequence according to the present invention when present in the organism.
[00226] Suitable organisms may include a prokaryote, fungus, yeast or a plant.
[00227] The term "transgenic organism" in relation to the present invention includes any organism that comprises the nucleotide sequence coding for the polypeptide according to the present invention and/or the products obtained therefrom, and/or wherein a promoter can allow expression of the nucleotide sequence according to the present invention within the organism.
Preferably the nucleotide sequence is incorporated in the genome of the organism.
Preferably the nucleotide sequence is incorporated in the genome of the organism.
[00228] The term "transgenic organism" does not cover native nucleotide coding sequences in their natural environment when they are under the control of their native promoter which is also in its natural environment.
[00229] Therefore, the transgenic organism of the present invention includes an organism comprising any one of, or combinations of, the nucleotide sequence coding for the polypeptide according to the present invention, constructs according to the present invention, vectors according to the present invention, plasmids according to the present invention, cells according to the present invention, tissues according to the present invention, or the products thereof.
[00230] For example, the transgenic organism may also comprise the nucleotide sequence coding for the polypeptide of the present invention under the control of a heterologous promoter.
Transformation of host cells/organisms
Transformation of host cells/organisms
[00231] As indicated earlier, the host organism can be a prokaryotic or a eukaryotic organism. Examples of suitable prokaryotic hosts include E. coil and Bacillus subtilis.
[00232] Teachings on the transformation of prokaryotic hosts are well documented in the art, for example see Sambrook et at (supra). If a prokaryotic host is used, then the nucleotide sequence may need to be suitably modified before transformation - such as by removal of introns.
[00233] Filamentous fungi cells may be transformed using various methods known in the art ¨ such as a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known. The use of Aspergillus as a host microorganism is described in EP 0 238 023.
[00234] Another host organism can be a plant. A review of the general techniques used for transforming plants may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mot Biol, 1991, 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27).
Further teachings on plant transformation may be found in EP-A-0449375.
Further teachings on plant transformation may be found in EP-A-0449375.
[00235] General teachings on the transformation of fungi, yeasts and plants are presented in following sections.
Transformed fungus
Transformed fungus
[00236] A host organism may be a fungus - such as a mold. Examples of suitable such hosts include any member belonging to the genera Thermomyces, Acremonium, Aspergillus, Penicillium, Mucor, Neurospora, Trichoderma and the like.
[00237] In one embodiment, the host organism may be a filamentous fungus.
[00238] Transforming filamentous fungi is discussed in US-A-5741665 which states that standard techniques for transformation of filamentous fungi and culturing the fungi are well known in the art. An extensive review of techniques as applied to N. crassa is found, for example in Davis and de Serres, Methods Enzymol (1971) 17A: 79-143.
[00239] Further teachings which may also be utilized in transforming filamentous fungi are reviewed in US-A-5674707.
[00240] In addition, gene expression in filamentous fungi is taught in in Punt et al. (2002) Trends Biotechnol 2002 May;20(5):200-6, Archer & Peberdy, Crit Rev Biotechnol (1997) 17(4):273-306.
[00241] The present invention encompasses the production of transgenic filamentous fungi according to the present invention prepared by use of these standard techniques.
[00242] In one aspect, the host organism can be of the genus Aspergillus, such as Aspergillus niger. .
[00243] A transgenic Aspergillus according to the present invention can also be prepared by following, for example, the teachings of Turner, G. 1994 (Vectors for genetic manipulation.
In: Martinelli S.D., Kinghorn J.R.(Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 29. Elsevier Amsterdam 1994. pp. 641-666).
In: Martinelli S.D., Kinghorn J.R.(Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 29. Elsevier Amsterdam 1994. pp. 641-666).
[00244] In one aspect, the host organism can be of the genus Trichoderma, such as Trichoderma Reesei.
Transformed yeast
Transformed yeast
[00245] In another embodiment, the transgenic organism can be a yeast.
[00246] A review of the principles of heterologous gene expression in yeast are provided in, for example, Methods Mot Blot (1995), 49:341-54, and Curr Opin Biotechnol (1997) Oct;8(5):554-60
[00247] In this regard, yeast ¨ such as the species Saccharomyces cerevisae or Pichia pastoris (see FEMS Microbiol Rev (2000 24(1):45-66), may be used as a vehicle for heterologous gene expression.
[00248] A review of the principles of heterologous gene expression in Saccharomyces cerevisiae and secretion of gene products is given by E Hinchcliffe E Kenny (1993, "Yeast as a vehicle for the expression of heterologous genes", Yeasts, Vol 5, Anthony H
Rose and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).
Rose and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).
[00249] For the transformation of yeast, several transformation protocols have been developed. For example, a transgenic Saccharomyces according to the present invention can be prepared by following the teachings of Hinnen et al., (1978, PNAS USA 75, 1929); Beggs, J D
(1978, Nature, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).
(1978, Nature, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).
[00250] The transformed yeast cells may be selected using various selective markers ¨ such as auxotrophic markers dominant antibiotic resistance markers.
Culturing and production
Culturing and production
[00251] Host cells transformed with the nucleotide sequence of the present invention may be cultured under conditions conducive to the production of the encoded polypeptide and which facilitate recovery of the polypeptide from the cells and/or culture medium.
[00252] The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in questions and obtaining expression of the polypeptide.
[00253] The protein produced by a recombinant cell may be displayed on the surface of the cell.
[00254] The protein may be secreted from the host cells and may conveniently be recovered from the culture medium using well-known procedures.
Secretion
Secretion
[00255] Often, it is desirable for the protein to be secreted from the expression host into the culture medium from where the protein may be more easily recovered. According to the present invention, the secretion leader sequence may be selected on the basis of the desired expression host. Hybrid signal sequences may also be used with the context of the present invention.
[00256] Typical examples of heterologous secretion leader sequences are those originating from the fungal amyloglucosidase (AG) gene (glaA - both 18 and 24 amino acid versions e.g.
from Aspergillus), the a-factor gene (yeasts e.g. Saccharomyces, Kluyveromyces and Hansenula) or the a-amylase gene (Bacillus).
from Aspergillus), the a-factor gene (yeasts e.g. Saccharomyces, Kluyveromyces and Hansenula) or the a-amylase gene (Bacillus).
[00257] By way of example, the secretion of heterologous proteins in E.
coil is reviewed in Methods Enzymol (1990) 182:132-43.
Detection
coil is reviewed in Methods Enzymol (1990) 182:132-43.
Detection
[00258] A variety of protocols for detecting and measuring the expression of the amino acid sequence are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (MA) and fluorescent activated cell sorting (FACS).
[00259] A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic and amino acid assays.
[00260] A number of companies such as Pharmacia Biotech (Piscataway, NJ), Promega (Madison, WI), and US Biochemical Corp (Cleveland, OH) supply commercial kits and protocols for these procedures.
[00261] Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US 3,817,837;
US ,850,752; US 3,939,350; US 3,996,345; US 4,277,437; US 4,275,149 and US
4,366,241.
US ,850,752; US 3,939,350; US 3,996,345; US 4,277,437; US 4,275,149 and US
4,366,241.
[00262] Also, recombinant immunoglobulins may be produced as shown in US
4,816,567.
Fusion proteins
4,816,567.
Fusion proteins
[00263] The amino acid sequence for use according to the present invention may be produced as a fusion protein, for example to aid in extraction and purification. Examples of fusion protein partners include Glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and (I3-galactosidase). It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences.
[00264] Preferably, the fusion protein will not hinder the activity of the protein sequence.
[00265] Gene fusion expression systems in E. colt have been reviewed in Curr Opin Biotechnol (1995) 6(5):501-6.
[00266] In another embodiment of the invention, the amino acid sequence may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a chimeric substance expressing a heterologous epitope that is recognized by a commercially available antibody.
Additional Proteins of interest (POIs)
Additional Proteins of interest (POIs)
[00267] The sequences for use according to the present invention may also be used in conjunction with one or more additional proteins of interest (POIs) or nucleotide sequences of interest (NOIs).
[00268] Non-limiting examples of POIs include: proteins or enzymes involved in starch metabolism, proteins or enzymes involved in glycogen metabolism, acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carboxypeptidases, catalases, cellulases, chitinases, chymosin, cutinase, deoxyribonucleases, epimerases, esterases, a-galactosidases,13-galactosidases, a-glucanases, glucan lysases, endo-13-glucanases, glucoamylases, glucose oxidases, a-glucosidases,13-glucosidases, glucuronidases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, tripeptidyl exopeptidases, laccases, lipases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, peroxidases, phenoloxidases, phytases, polygalacturonases, proline endoproteases, rhamno-galacturonases, ribonucleases, thaumatin, transferases, exoproteases, transport proteins, transglutaminases, aminopeptidases, hexose oxidase (D-hexose: 02-oxidoreductase, EC 1.1.3.5) or combinations thereof The NOT
may even be an antisense sequence for any of those sequences.
may even be an antisense sequence for any of those sequences.
[00269] The POI may even be a fusion protein, for example to aid in extraction and purification.
[00270] The POI may even be fused to a secretion sequence.
[00271] Other sequences can also facilitate secretion or increase the yield of secreted POI.
Such sequences could code for chaperone proteins as for example the product of Aspergillus niger cyp B gene described in UK patent application 9821198Ø
Such sequences could code for chaperone proteins as for example the product of Aspergillus niger cyp B gene described in UK patent application 9821198Ø
[00272] The NOT may be engineered in order to alter their activity for a number of reasons, including but not limited to, alterations which modify the processing and/or expression of the expression product thereof By way of further example, the NOT may also be modified to optimize expression in a particular host cell. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites.
[00273] The NOT may include within it synthetic or modified nucleotides¨
such as methylphosphonate and phosphorothioate backbones.
such as methylphosphonate and phosphorothioate backbones.
[00274] The NOT may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' 0-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
General recombinant DNA methodology techniques
General recombinant DNA methodology techniques
[00275] The present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art.
Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et at. (1995 and periodic supplements;
Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B.
Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons;
M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, D. M.
J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA in Methods in Enzymology, Academic Press. Each of these general texts is herein incorporated by reference.
EXAMPLES
Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et at. (1995 and periodic supplements;
Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B.
Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons;
M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, D. M.
J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA in Methods in Enzymology, Academic Press. Each of these general texts is herein incorporated by reference.
EXAMPLES
[00276] The present disclosure is described in further detail in the following examples, which are not in any way intended to limit the scope of the disclosure as claimed. The attached figures are meant to be considered as integral parts of the specification and description of the disclosure. The following examples are offered to illustrate, but not to limit the claimed disclosure.
Example 1 ¨ Cloning and transformation of TRI031, TRI032, TRI033, TRI034, TRI035, TRI036, TRI037, TRI038
Example 1 ¨ Cloning and transformation of TRI031, TRI032, TRI033, TRI034, TRI035, TRI036, TRI037, TRI038
[00277] Synthetic genes (TRI031, TRI032, TRI033, TRI034, TRI035, TRI036, TRI037, TRI038) encoding fungal aminopeptidases type-2 (pepN 2, belonging to Merops family M28.008.; merops.sanger.ac.uk/) were ordered from Geneart (Life Technologies) as codon-optimized genes for expression in Trichoderma reesei. TRI031 corresponds to NCBI accession number: XP 001258675 from Neosartorya fischeri NRRL 181; TRI032 corresponds to NCBI
accession number: XP 003667354 from Myceliophthora thermophila ATCC 42464;
corresponds to NCBI accession number: EGU74500 from Fusarium oxysporum Fo5176;
corresponds to NCBI accession number: ENH69875from Fusarium oxysporum f. sp.
cubense race 1; TRI035 corresponds to NCBI accession number: XP 001273779 from Aspergillus clavatus NRRL 1; TRI036 corresponds to NCBI accession number: EG523402 Chaetomium thermophilum var. thermophilum DSM 1495; TRI037 corresponds to NCBI accession number:
XP 001217759 from Aspergillus terreus NIH2624; and TRI038 corresponds to NCBI
accession number: XP 681714 from Aspergillus nidulans FGSC A4.
accession number: XP 003667354 from Myceliophthora thermophila ATCC 42464;
corresponds to NCBI accession number: EGU74500 from Fusarium oxysporum Fo5176;
corresponds to NCBI accession number: ENH69875from Fusarium oxysporum f. sp.
cubense race 1; TRI035 corresponds to NCBI accession number: XP 001273779 from Aspergillus clavatus NRRL 1; TRI036 corresponds to NCBI accession number: EG523402 Chaetomium thermophilum var. thermophilum DSM 1495; TRI037 corresponds to NCBI accession number:
XP 001217759 from Aspergillus terreus NIH2624; and TRI038 corresponds to NCBI
accession number: XP 681714 from Aspergillus nidulans FGSC A4.
[00278] The genes were ordered with Gateway-specific recombination sites (attB1 and attB2) (Life Technologies) flanking the coding region and delivered as plasmid stocks in the pDonr221 Gateway vector (Life Technologies). The TRI031, TRI032, TRI033, TRI034, TRI035, TRI036, TRI037, TRI038 amino acid sequences have predicted secretion signal sequences (SignalP 4.0: discriminating signal peptides from transmembrane regions.
Thomas Nordahl Petersen, Soren Brunak, Gunnar von Heijne & Henrik Nielsen. Nature Methods, 8:785-786, 2011) and these endogenous signal sequences were replaced by a leader sequence containing a Kozak sequence, the secretion signal sequence from the Trichoderma reesei acidic fungal protease (AFP) and an intron from a Trichoderma reesei glucoamylase gene (TrGA1) (see Figure 1).
Thomas Nordahl Petersen, Soren Brunak, Gunnar von Heijne & Henrik Nielsen. Nature Methods, 8:785-786, 2011) and these endogenous signal sequences were replaced by a leader sequence containing a Kozak sequence, the secretion signal sequence from the Trichoderma reesei acidic fungal protease (AFP) and an intron from a Trichoderma reesei glucoamylase gene (TrGA1) (see Figure 1).
[00279] The synthetic genes in the pDonr221 vector was recombined into the destination vector pTrex8gM using LR CLONASETM enzyme mix (Life Technologies) resulting in the expression vectors pTrex8gM TRI031 (SEQ ID NO: 9), pTrex8gM TRI032 (SEQ ID NO:
10), pTrex8gM TRI033 (SEQ ID NO: 11), pTrex8gM TRI034 (SEQ ID NO: 12), pTrex8gM TRI035 (SEQ ID NO: 13), pTrex8gM TRI036 (SEQ ID NO: 14), pTrex8gM TRI037 (SEQ ID NO: 15), and pTrex8gM TRI038 (SEQ ID NO: 16).
10), pTrex8gM TRI033 (SEQ ID NO: 11), pTrex8gM TRI034 (SEQ ID NO: 12), pTrex8gM TRI035 (SEQ ID NO: 13), pTrex8gM TRI036 (SEQ ID NO: 14), pTrex8gM TRI037 (SEQ ID NO: 15), and pTrex8gM TRI038 (SEQ ID NO: 16).
[00280] 1.5-17 tg of the expression vectors were transformed individually into a Trichoderma reesei strain CellulightTM using PEG mediated protoplast transformation essentially as described in (US 8,592,194 B2). PEG-Protoplast method with slight modifications was used for transformation, as indicated. For protoplasts preparation, spores were grown for approximately 18 hours at 26 C in Trichoderma germination medium with 10 mM
uridine to complement the pyr auxotrophy. (Trichoderma germination medium: 40 ml 50%
glucose, 2 g/L
peptone, 15g/L KH2PO4, 5g/L (NH4)2504, 2.4 ml 1M Mg504, 4.1 ml 1M CaC12, 1 ml of 400X T
reesei Trace elements solution {200 g/L FeSO4x7H20, 16 g/L ZnSO4x7H20, 1.4 g/L
MnSO4xH20, 3.2 g/L CuSO4x5H20, 0.8 g/L H3B03, 175 g/L citric acid}) at shaking speed of 200 rpm. Germinating spores were harvested by centrifugation, washed and treated with 45 mg/ml of lysing enzyme solution (Trichoderma harzianum, Sigma cat# L1412) to lyse the fungal cell walls. Further preparation of protoplasts was performed by a standard method, as described by Penttila et at. (Gene 61(1987) 155-164).
uridine to complement the pyr auxotrophy. (Trichoderma germination medium: 40 ml 50%
glucose, 2 g/L
peptone, 15g/L KH2PO4, 5g/L (NH4)2504, 2.4 ml 1M Mg504, 4.1 ml 1M CaC12, 1 ml of 400X T
reesei Trace elements solution {200 g/L FeSO4x7H20, 16 g/L ZnSO4x7H20, 1.4 g/L
MnSO4xH20, 3.2 g/L CuSO4x5H20, 0.8 g/L H3B03, 175 g/L citric acid}) at shaking speed of 200 rpm. Germinating spores were harvested by centrifugation, washed and treated with 45 mg/ml of lysing enzyme solution (Trichoderma harzianum, Sigma cat# L1412) to lyse the fungal cell walls. Further preparation of protoplasts was performed by a standard method, as described by Penttila et at. (Gene 61(1987) 155-164).
[00281] In general, transformation mixtures containing 1.5-17 tg of DNA and ¨5x 107 protoplasts in a total volume of 200 !IL were treated with 2 mL of 25% PEG
solution, diluted with 2 volumes of 1.2M sorbito1/10mM Tris, pH7.5/ 10mM CaC12 solution, mixed with 3%
selective top agarose, 1 M sorbitol, 10 mM NH4C1, lx MINI solution ( 2x MINI
solution: 30 g/L
KH2PO4, 20 mL 1M acetamide, 20 ml 1M CsCl, 6 ml 20% MgSO4x7H20, 6 ml 20%
CaC12x2H20, 2 mL T reesei Trace elements solution (400x), 80 mL 50% glucose, make up to 1 liter, pH 4.5 ) and poured on to MM plates with 10 mM NH4C1 (MM plates 2%
agar, lx Mlvi solution). Transformants were selected for prototrophic growth on MINI plates (lacking uridine).
Plates were incubated for 5-7 days at 28 C until sporulation occurred. Stable looking transformants were transferred to new MINI plates with 10 mM NH4C1 for better sporulation.
When sporulated, spores were harvested using a solution of 085% NaC1, 0015%
'MEIN 80 Spore suspensions were used to inoculate liquid cultures either in a 24-well MTP format (for screening) or shake flasks (for validation studies). Preculture in 3 mL YEG
medium (5 g/L yeast extract, 22g/L glucose, H20) Main culture in the following production medium (Production medium: 35 g/L 61% glucose/sophorose mix, 9 g/L casmino acids, 5 g/L
(NH4)2504, 4.5 g/L
KH2PO4, 1 g/L CaC12x2H20, 1 g/L MgSO4x7H20, 33 g/L PIPPS buffer, pH 5.5, 2.5 mL/L of 400X T reesei trace elements (175 g/L citric acid, 200 g/L FeSO4x7H20, 16 g/L
ZnSO4x7H20, 3.2 g/L CuSO4x5H20, 1.4 g/L MnSO4xH20, 0.8 g/L boric acid) pH 5.5. 3 mL of production medium was added to produce variants in 24-well MTPs. For shake flasks, volumes were scaled up.
solution, diluted with 2 volumes of 1.2M sorbito1/10mM Tris, pH7.5/ 10mM CaC12 solution, mixed with 3%
selective top agarose, 1 M sorbitol, 10 mM NH4C1, lx MINI solution ( 2x MINI
solution: 30 g/L
KH2PO4, 20 mL 1M acetamide, 20 ml 1M CsCl, 6 ml 20% MgSO4x7H20, 6 ml 20%
CaC12x2H20, 2 mL T reesei Trace elements solution (400x), 80 mL 50% glucose, make up to 1 liter, pH 4.5 ) and poured on to MM plates with 10 mM NH4C1 (MM plates 2%
agar, lx Mlvi solution). Transformants were selected for prototrophic growth on MINI plates (lacking uridine).
Plates were incubated for 5-7 days at 28 C until sporulation occurred. Stable looking transformants were transferred to new MINI plates with 10 mM NH4C1 for better sporulation.
When sporulated, spores were harvested using a solution of 085% NaC1, 0015%
'MEIN 80 Spore suspensions were used to inoculate liquid cultures either in a 24-well MTP format (for screening) or shake flasks (for validation studies). Preculture in 3 mL YEG
medium (5 g/L yeast extract, 22g/L glucose, H20) Main culture in the following production medium (Production medium: 35 g/L 61% glucose/sophorose mix, 9 g/L casmino acids, 5 g/L
(NH4)2504, 4.5 g/L
KH2PO4, 1 g/L CaC12x2H20, 1 g/L MgSO4x7H20, 33 g/L PIPPS buffer, pH 5.5, 2.5 mL/L of 400X T reesei trace elements (175 g/L citric acid, 200 g/L FeSO4x7H20, 16 g/L
ZnSO4x7H20, 3.2 g/L CuSO4x5H20, 1.4 g/L MnSO4xH20, 0.8 g/L boric acid) pH 5.5. 3 mL of production medium was added to produce variants in 24-well MTPs. For shake flasks, volumes were scaled up.
[00282] Cultures were grown for 7 days at 28 C and 80% humidity with shaking at 180 rpm. Culture supernatants were harvested by vacuum filtration and used to assay their performance as well as expression level.
[00283] The amino acid and nucleotide sequences for the pepN 2 enzymes are depicted below:
[00284] SEQ ID NO: 1 = Amino acid sequence of TRI031. Underlined is the secretion signal encoded by the leader sequence described in figure 1:
MQTFGAFLVSFLAASGLAAANGPGWDWKPPVHPKVLPQMIHLWDLMHGAQKLEDFAYAYPERNRVFGG
PAHEDTVNYLYRELKKTGYYDVYKQPQVHQWTRADQALTVDGKSYVATTMTYSPSVNVTAPLAVVNNL
GCVESDYPADLKGKIALVSRGECPFATKSVLSAKAGAAAALVYNNIEGSMAGTLGGPTSELGPYAPIAGISL
AD GQALIQMIQAGTVTANLWID SKVENRTTYNVIAQTKGGDPNNVVAL GGHTD SVEAGPGINDD GS GIISN
LVVAKALTRFSVKNAVRFCFWTAEEFGLLGSSYYVNSLNA __________________________________ IEKAKIRLYLNFDMIASPNYALMIYD GD GS
AFNLTGPAGSAQIERLFEDYYKSIRKPFVPTEFNGRSDYEAFILNGIPAGGIFTGAEAIK ______________ IEEQAKLFGGQAG
VALDANYHAKGDNMTNLNREAFLINSKATAFAVATYANSLD SIP SRNMSTVVKRSQLEQAKKS TPHTHTG
GTGCYKDRVEQ (Seq ID 110 1) The sequence absent the underlined leader sequence is denoted SEQ ID NO: 18.
MQTFGAFLVSFLAASGLAAANGPGWDWKPPVHPKVLPQMIHLWDLMHGAQKLEDFAYAYPERNRVFGG
PAHEDTVNYLYRELKKTGYYDVYKQPQVHQWTRADQALTVDGKSYVATTMTYSPSVNVTAPLAVVNNL
GCVESDYPADLKGKIALVSRGECPFATKSVLSAKAGAAAALVYNNIEGSMAGTLGGPTSELGPYAPIAGISL
AD GQALIQMIQAGTVTANLWID SKVENRTTYNVIAQTKGGDPNNVVAL GGHTD SVEAGPGINDD GS GIISN
LVVAKALTRFSVKNAVRFCFWTAEEFGLLGSSYYVNSLNA __________________________________ IEKAKIRLYLNFDMIASPNYALMIYD GD GS
AFNLTGPAGSAQIERLFEDYYKSIRKPFVPTEFNGRSDYEAFILNGIPAGGIFTGAEAIK ______________ IEEQAKLFGGQAG
VALDANYHAKGDNMTNLNREAFLINSKATAFAVATYANSLD SIP SRNMSTVVKRSQLEQAKKS TPHTHTG
GTGCYKDRVEQ (Seq ID 110 1) The sequence absent the underlined leader sequence is denoted SEQ ID NO: 18.
[00285] SEQ ID NO: 2 = Amino acid sequence of TRI032. Underlined is the secretion signal encoded by the leader sequence described in figure 1.
MQTF GAFL VSFL AA S GL AAAGGH GG S SGLGCD S QRPL VS
SEKLQSLIKKEDLLAGSQELQDIATAHGGHRA
FGS S GHNATVDFLYYTLKALDYYNVTKQPFKEIFS S GTG SL TVD GED IEAETL TYTP S
GSATDKPVVVVAN
VGCDAADYP AEVAGNIALIKRGTCTF SQKSVNAKAAGAVAAIIYNNAEGKL SGTL GQPFLDYAPVLGITLE
AGEALLAKLAGGPVTATLQIDALVEERVTYNVIAETKEGDHSNVLVLGGHTD SVPAGPGINDD GS GTIGML
TVAKALTKFRVKNAVRFAFW SAEEYGLLGSYAYIK SINS SAAEL SKIRAYLNFDMIASPNYIYGIYDGDGNA
FNLTGPAGSDVIERNFENFFKRKHTPSVPTEFSGRSDYAAFIENGIPSGGLFTGAEVLK ________________ lEREAELFGGRAGV
AYDVNYHQAGDTVDNLALDAFLLNTKAIAD SVATYAL SFD GLPRVD GKKRRWD AHRARMLKRS A G SH G
HAHLHSGPCGGGASI (Seq ID 110 2) The sequence absent the underlined leader sequence is denoted SEQ ID NO: 19.
MQTF GAFL VSFL AA S GL AAAGGH GG S SGLGCD S QRPL VS
SEKLQSLIKKEDLLAGSQELQDIATAHGGHRA
FGS S GHNATVDFLYYTLKALDYYNVTKQPFKEIFS S GTG SL TVD GED IEAETL TYTP S
GSATDKPVVVVAN
VGCDAADYP AEVAGNIALIKRGTCTF SQKSVNAKAAGAVAAIIYNNAEGKL SGTL GQPFLDYAPVLGITLE
AGEALLAKLAGGPVTATLQIDALVEERVTYNVIAETKEGDHSNVLVLGGHTD SVPAGPGINDD GS GTIGML
TVAKALTKFRVKNAVRFAFW SAEEYGLLGSYAYIK SINS SAAEL SKIRAYLNFDMIASPNYIYGIYDGDGNA
FNLTGPAGSDVIERNFENFFKRKHTPSVPTEFSGRSDYAAFIENGIPSGGLFTGAEVLK ________________ lEREAELFGGRAGV
AYDVNYHQAGDTVDNLALDAFLLNTKAIAD SVATYAL SFD GLPRVD GKKRRWD AHRARMLKRS A G SH G
HAHLHSGPCGGGASI (Seq ID 110 2) The sequence absent the underlined leader sequence is denoted SEQ ID NO: 19.
[00286] SEQ ID NO: 3 = Amino acid sequence of TRI033. Underlined is the secretion signal encoded by the leader sequence described in figure 1.
MQTFGAFLVSFLAASGLAAATKKPLVNELKLQKDINIKDLMAGAQKLQDIAEANGNTRVFGGAGHNATV
DYLYKTLKATGYYNVKKQPFTELYSAGTASLKVDGDDITAAIMTYTPAGEATGPLVVAENLGCEASDFPA
ESEGKVVLVLRGECPFSQKSTNGKTAGAAAVIVYNNVPGELAGTL GEPFGEFAPIVGISQEDGQAILAKTKA
GEVTVDLKVDATVENRVTFNVIAETKEGDHDNVLVVGGH SD SVAAGPGINDD GS GIIGILKVAQALTKYR
VKNAVRFGFWSAEEFGLL GSYAYMKS INGSDAEVAKIRAYLNFDMIASPNYVYGIYD GD GSAFNLTGPAG
SDAIEKDFERFFKTKRLGYVP SEF S GRSDYAAFIENGIP S GGLFTGAEQLK ___________________ lEEEAKKFGGEAGVAYDINYH
KIGDDINNLNKEAFL VNTQ AIANSVARYAKTWKSLPKVTHNTRRWDAEVASVLKRS SGHSHAGGPCGSVS
V (Seq ID no 3) The sequence absent the underlined leader sequence is denoted SEQ ID NO: 20.
MQTFGAFLVSFLAASGLAAATKKPLVNELKLQKDINIKDLMAGAQKLQDIAEANGNTRVFGGAGHNATV
DYLYKTLKATGYYNVKKQPFTELYSAGTASLKVDGDDITAAIMTYTPAGEATGPLVVAENLGCEASDFPA
ESEGKVVLVLRGECPFSQKSTNGKTAGAAAVIVYNNVPGELAGTL GEPFGEFAPIVGISQEDGQAILAKTKA
GEVTVDLKVDATVENRVTFNVIAETKEGDHDNVLVVGGH SD SVAAGPGINDD GS GIIGILKVAQALTKYR
VKNAVRFGFWSAEEFGLL GSYAYMKS INGSDAEVAKIRAYLNFDMIASPNYVYGIYD GD GSAFNLTGPAG
SDAIEKDFERFFKTKRLGYVP SEF S GRSDYAAFIENGIP S GGLFTGAEQLK ___________________ lEEEAKKFGGEAGVAYDINYH
KIGDDINNLNKEAFL VNTQ AIANSVARYAKTWKSLPKVTHNTRRWDAEVASVLKRS SGHSHAGGPCGSVS
V (Seq ID no 3) The sequence absent the underlined leader sequence is denoted SEQ ID NO: 20.
[00287] SEQ ID NO: 4 = Amino acid sequence of TRI034. Underlined is the secretion signal encoded by the leader sequence described in figure 1.
MQTFGAFLVSFLAASGLAAALQIPLNLQVPKL SWNLFGDDLPLVDTKELQKSIKPENLEARAKDLYEIAKN
GEEEYGHPTRVIGSEGHLGTL SYIHAELAKL GGYYSVSNQQFPAVSGNVFESRLVIGD SVPKQASPMGLTPP
TKNKEPVHGTLVLVDNEGCDASDYPEAVKGNIALILRGTCPFGTKS GNAGKAGAVAAVVYNYEKDEVHG
TL GTP SPDHVATFGLGGEEGKAVAKKLKD GEKVDAIAYID AEVKTI STTNIIAQ TRGGDPDNCVML GGH
SD
SVAEGPGINDD GS G SI SVLEVAVQL TKYRVNNCVRF AWWAAEEEGLL GSDHYVSVLPEDENRKIRLFMDY
DMMASPNFAYQIYNATNAENPKGSEELRDLYVNWYEEQ GLNYTFIPFDGRSDYDGFIRGGIPAGGIATGAE
TYSDKTKYHGSKLFI (Seq ID no 4) The sequence absent the underlined leader sequence is denoted SEQ ID NO: 21.
MQTFGAFLVSFLAASGLAAALQIPLNLQVPKL SWNLFGDDLPLVDTKELQKSIKPENLEARAKDLYEIAKN
GEEEYGHPTRVIGSEGHLGTL SYIHAELAKL GGYYSVSNQQFPAVSGNVFESRLVIGD SVPKQASPMGLTPP
TKNKEPVHGTLVLVDNEGCDASDYPEAVKGNIALILRGTCPFGTKS GNAGKAGAVAAVVYNYEKDEVHG
TL GTP SPDHVATFGLGGEEGKAVAKKLKD GEKVDAIAYID AEVKTI STTNIIAQ TRGGDPDNCVML GGH
SD
SVAEGPGINDD GS G SI SVLEVAVQL TKYRVNNCVRF AWWAAEEEGLL GSDHYVSVLPEDENRKIRLFMDY
DMMASPNFAYQIYNATNAENPKGSEELRDLYVNWYEEQ GLNYTFIPFDGRSDYDGFIRGGIPAGGIATGAE
TYSDKTKYHGSKLFI (Seq ID no 4) The sequence absent the underlined leader sequence is denoted SEQ ID NO: 21.
[00288] SEQ ID NO: 5 = Amino acid sequence of TRI035. Underlined is the secretion signal encoded by the leader sequence described in figure 1.
MQTFGAFLVSFLAASGLAAANAPGGPGGHGRKLPVNPKTFPNEIRLKDLLHGSQKLEDFAYAYPERNRVF
GGQAHLDTVNYLYRELKKTGYYDVYKQPQVHQWTRADQ SLTLGGD S IQA S TMTY SP SVNVTAPL SLVSK
LGCAEGDYSADVKGKIALVSRGECSFAQKSVL SAKAGAVATIVYNNVDGSLAGTLGGATSEL GPYSPIIGIT
LAAGQDLVARLQAAP ____________________________________________________________ I
EVSLWID SKVENRTTYNVIAQTKGGDPNNVVAL GGHTD SVENGPGINDDGS GVI
SNLVVAKALTRYSVKNAVRFCFWTAEEFGLLGSNYYVDNL SP AEL AKIRLYLNFDMIASPNYALMIYD GD
GSAFNLTGPPGSAQIESLFENYYKSIKQGFVPTAFDGRSDYEGFILKGIPAGGVFTGAESLKTEEQARLF GGQ
AGVALDANYHAKGDNMINLNHKAFLINSRATAFAVATYANNL S SIPPRNATVVKRESMKWTKREEPHTH
GADTGCFASRVKE (Seq ID 110 5) The sequence absent the underlined leader sequence is denoted SEQ ID NO: 22.
MQTFGAFLVSFLAASGLAAANAPGGPGGHGRKLPVNPKTFPNEIRLKDLLHGSQKLEDFAYAYPERNRVF
GGQAHLDTVNYLYRELKKTGYYDVYKQPQVHQWTRADQ SLTLGGD S IQA S TMTY SP SVNVTAPL SLVSK
LGCAEGDYSADVKGKIALVSRGECSFAQKSVL SAKAGAVATIVYNNVDGSLAGTLGGATSEL GPYSPIIGIT
LAAGQDLVARLQAAP ____________________________________________________________ I
EVSLWID SKVENRTTYNVIAQTKGGDPNNVVAL GGHTD SVENGPGINDDGS GVI
SNLVVAKALTRYSVKNAVRFCFWTAEEFGLLGSNYYVDNL SP AEL AKIRLYLNFDMIASPNYALMIYD GD
GSAFNLTGPPGSAQIESLFENYYKSIKQGFVPTAFDGRSDYEGFILKGIPAGGVFTGAESLKTEEQARLF GGQ
AGVALDANYHAKGDNMINLNHKAFLINSRATAFAVATYANNL S SIPPRNATVVKRESMKWTKREEPHTH
GADTGCFASRVKE (Seq ID 110 5) The sequence absent the underlined leader sequence is denoted SEQ ID NO: 22.
[00289] SEQ ID NO: 6 = Amino acid sequence of TRI036. Underlined is the secretion signal encoded by the leader sequence described in figure 1.
MQTFGAFLVSFLAASGLAAAGGPHGFGLPKIDLRPMVS SNRLQSMITLKDLMDGAKKLQDIATKNGGNRA
FGGAGHNATVDYLYKTLT SL GGYYTVKKQPFKEIF S SGS GSLIVD GQGIDAGIMTYTP GGSATANLVQ
VAN
LGCEDEDYPAEVAGNIALISRGSCTFS SK SLKAKAAGAVGAIVYNNVP GEL SGTL GTPFLDYAPIVGI S
QED
GQVILEKLAAGPVTATLNIDAIVEERTTYNVIAETKEGDHNNVL IVGGH SD S VAAGP GINDD GS
GTIGILTV
AKALAKANVRIKNAVRFAFW SAEEFGLL GSYAYMKSLNESEAEVAKIRAYLNFDMIASPNYIYGIYDGDG
NAFNLTGPAGSDIIEKDFEDFFKKKKTP S VP __________________________________________ IEFSGRSDYAAFIENGIP SGGLFTGAEVLKTEEEAKLFGGKA
GVAYD VNYHKAGDTVDNLAKD AFLLNTKAIANS VAKYAASWAGFPKP SAVRRRYDADMAQLLKRSGGV
HGHGPHTHSGPCGGGDLL (Seq ID no 6) The sequence absent the underlined leader sequence is denoted SEQ ID NO: 23.
MQTFGAFLVSFLAASGLAAAGGPHGFGLPKIDLRPMVS SNRLQSMITLKDLMDGAKKLQDIATKNGGNRA
FGGAGHNATVDYLYKTLT SL GGYYTVKKQPFKEIF S SGS GSLIVD GQGIDAGIMTYTP GGSATANLVQ
VAN
LGCEDEDYPAEVAGNIALISRGSCTFS SK SLKAKAAGAVGAIVYNNVP GEL SGTL GTPFLDYAPIVGI S
QED
GQVILEKLAAGPVTATLNIDAIVEERTTYNVIAETKEGDHNNVL IVGGH SD S VAAGP GINDD GS
GTIGILTV
AKALAKANVRIKNAVRFAFW SAEEFGLL GSYAYMKSLNESEAEVAKIRAYLNFDMIASPNYIYGIYDGDG
NAFNLTGPAGSDIIEKDFEDFFKKKKTP S VP __________________________________________ IEFSGRSDYAAFIENGIP SGGLFTGAEVLKTEEEAKLFGGKA
GVAYD VNYHKAGDTVDNLAKD AFLLNTKAIANS VAKYAASWAGFPKP SAVRRRYDADMAQLLKRSGGV
HGHGPHTHSGPCGGGDLL (Seq ID no 6) The sequence absent the underlined leader sequence is denoted SEQ ID NO: 23.
[00290] SEQ ID NO: 7 = Amino acid sequence of TRI037. Underlined is the secretion signal encoded by the leader sequence described in figure 1.
MQTFGAFLVSFLAASGLAAAEGL GNHGRKLDPNKFTKDIKLKDLLKGSQKLEDFAYAYPERNRVFGGKAH
QDTVNWIYNELKKTGYYDVYKQPQVHLWSNAEQ SLTVDGEAIDATTMTYSPSLKETTAEVVVVPGLGCT
AADYPADVAGKIALIQRGSCTFGEKSVYAAAANAAAAIVYNNVDGSL SGTL GAATSELGPYAPIVGISLAD
GQNLVSLAQAGPLTVDLYINSQMENRTTHNVIAKSKGGDPNNVIVIGGHSDAVNQGPGVNDDGSGIISNLV
IAKALTKYSLKNSVTWAFWTAEEFGLLGSEFYVNSL SAAEKDKIKLYLNFDMIASPNYALMIYDGD GSTFN
MTGPAGSAEIEHLFEDYYKSRGL SYIPTAFD GRSDYEAFILNGIPAGGLFTGAEQIKTEEQVAMFGGQAGVA
YDPNYHAAGDNMTNL SEEAFLINSKATAFAVATYANSLESIPPRNATMSIQTRSASRRAAAHRRAAKPHSH
SGGTGCWHTRVEL (Seq ID no?) The sequence absent the underlined leader sequence is denoted SEQ ID NO: 24.
MQTFGAFLVSFLAASGLAAAEGL GNHGRKLDPNKFTKDIKLKDLLKGSQKLEDFAYAYPERNRVFGGKAH
QDTVNWIYNELKKTGYYDVYKQPQVHLWSNAEQ SLTVDGEAIDATTMTYSPSLKETTAEVVVVPGLGCT
AADYPADVAGKIALIQRGSCTFGEKSVYAAAANAAAAIVYNNVDGSL SGTL GAATSELGPYAPIVGISLAD
GQNLVSLAQAGPLTVDLYINSQMENRTTHNVIAKSKGGDPNNVIVIGGHSDAVNQGPGVNDDGSGIISNLV
IAKALTKYSLKNSVTWAFWTAEEFGLLGSEFYVNSL SAAEKDKIKLYLNFDMIASPNYALMIYDGD GSTFN
MTGPAGSAEIEHLFEDYYKSRGL SYIPTAFD GRSDYEAFILNGIPAGGLFTGAEQIKTEEQVAMFGGQAGVA
YDPNYHAAGDNMTNL SEEAFLINSKATAFAVATYANSLESIPPRNATMSIQTRSASRRAAAHRRAAKPHSH
SGGTGCWHTRVEL (Seq ID no?) The sequence absent the underlined leader sequence is denoted SEQ ID NO: 24.
[00291] SEQ ID NO: 8 = Amino acid sequence of TRI038. Underlined is the secretion signal encoded by the leader sequence described in figure 1.
MQTFGAFLVSFLAASGLAAAGKHKPLVTPEALQDLITLDDLLAGSQQLQDFAYAYPERNRVFGGRAHDDT
VNWLYRELKRTGYYHVYKQPQVHLYSNAEESLTVNGEAIEATTMTYSP S ANASAELAVI S GL GC SPADFA
SDVAGKVVLVQRGNCTFGEKSVYAAAADAAATIVYNNVEGSL SGTL GAAQ SEQGPYS GIVGI SL AD GEAL
LALAEEGPVHVDLWID SVMENRTTYNVIAQTKGGDPDNVVTLGGH SD SVEAGPGINDDGSGIISNLVIARA
LTKF S TKHAVRFFF WTAEEF GLL G SD YYVS SL
SPAELAKIRLYLNFDMIASPNYGLLLYDGDGSAFNLTGPA
GSDAIEKLFYDYFQSIGQATVETEFD GRSDYEAFILNGIPAGGVFTGAEEIKSEEEVALWGGEAGVAYD ANY
HQVGDTIDNLN __ lEAYLLNSKATAFAVATYANDLSTIPKREMTTAVKRANVNGHMHRRTMPKKRQTAHRH
AAKGCFHSRVEQ (Seq ID no 8) The sequence absent the underlined leader sequence is denoted SEQ ID NO: 25.
MQTFGAFLVSFLAASGLAAAGKHKPLVTPEALQDLITLDDLLAGSQQLQDFAYAYPERNRVFGGRAHDDT
VNWLYRELKRTGYYHVYKQPQVHLYSNAEESLTVNGEAIEATTMTYSP S ANASAELAVI S GL GC SPADFA
SDVAGKVVLVQRGNCTFGEKSVYAAAADAAATIVYNNVEGSL SGTL GAAQ SEQGPYS GIVGI SL AD GEAL
LALAEEGPVHVDLWID SVMENRTTYNVIAQTKGGDPDNVVTLGGH SD SVEAGPGINDDGSGIISNLVIARA
LTKF S TKHAVRFFF WTAEEF GLL G SD YYVS SL
SPAELAKIRLYLNFDMIASPNYGLLLYDGDGSAFNLTGPA
GSDAIEKLFYDYFQSIGQATVETEFD GRSDYEAFILNGIPAGGVFTGAEEIKSEEEVALWGGEAGVAYD ANY
HQVGDTIDNLN __ lEAYLLNSKATAFAVATYANDLSTIPKREMTTAVKRANVNGHMHRRTMPKKRQTAHRH
AAKGCFHSRVEQ (Seq ID no 8) The sequence absent the underlined leader sequence is denoted SEQ ID NO: 25.
[00292] SEQ ID NO: 9 = Nucleotide sequence of the pTrex8gM TRI031 expression construct.
CTAGAGTTGTGAAGTCGGTAATCCCGCTGTATAGTAATACGAGTCGCATCTAAATACTCCGAAGCTGCTGCGAACCCGG
AGAATC
GAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAA
TCATGG
CGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTCCCGAAAAAACTCGGAGATTCCTAAGT
AGCGAT
GGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAAC
TGTTCC
GTACCCCACCTCTTCTCAACCTTTGGCGTTTCCCTGATTCAGCGTACCCGTACAAGTCGTAATCACTATTAACCCAGAC
TGACCGG
ACGTGTTTTGCCCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGACCGACTGGGGCTGTTCGA
AGCCCGA
ATGTAGGATTGTTATCCGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGCCTCGAAGGTTCA
CGGCAA
GGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCCGCAATGCAGCATCACTGGAAAATACAAACCAATGGC
TAAAA
GTACATAAGTTAATGCCTAAAGAAGTCATATACCAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATT
TGCCAA
CGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGG
TGATCCC
CCAATTGGGTCGCTTGTTTGTTCCGGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTCGGAGCGTTTTGCATAC
AACCAA
GGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAG
GTTTGT
CTGCCGATACGACGAATACTGTATAGTCACTTCTGATGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCA
AAAGAT
TGAGTTGAAACTGCCTAAGATCTCGGGCCCTCGGGCCTTCGGCCTTTGGGTGTACATGTTTGTGCTCCGGGCAAATGCA
AAGTGTG
GTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGT
TTCGAA
TAGAAAGAGAAGCTTAGCCAAGAACAATAGCCGATAAAGATAGCCTCATTAAACGGAATGAGCTAGTAGGCAAAGTCAG
CGAAT
GTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGAC
TTGTACA
CCATCTTTTGAGGCACAGAAACCCAATAGTCAACCATCACAAGTTTGTACAAAAAAGCAGGCTTCACCATGCAGACCTT
CGGTGC
TTTTCTCGTTTCCTTCCTCGCCGCCAGGTAAGTTGGCCTTGATGAACCATATCATATATCGCCGAGAAGTGGACCGCGT
GCTGAGA
CTGAGACAGCGGCCTGGCCGCGGCCAACGGACCTGGATGGGATTGGAAGCCCCCCGTCCACCCCAAGGTCCTCCCCCAG
ATGATC
CACCTCTGGGACCTCATGCACGGCGCCCAGAAGCTCGAAGATTTCGCCTACGCCTACCCCGAGCGCAACCGCGTCTTTG
GCGGCC
CTGCCCACGAGGACACCGTCAACTACCTCTACCGCGAGCTGAAGAAGACCGGCTACTACGACGTCTACAAGCAGCCCCA
GGTCCA
CCAGTGGACCCGAGCCGATCAGGCCCTCACCGTCGACGGCAAGAGCTACGTCGCCACCACCATGACCTACAGCCCCAGC
GTCAAC
GTCACCGCCCCTCTCGCCGTCGTCAACAACCTCGGCTGCGTCGAGAGCGACTACCCCGCCGACCTCAAGGGCAAGATCG
CCCTCG
TTTCTCGCGGCGAGTGCCCCTTCGCCACCAAGTCTGTCCTCAGCGCCAAGGCTGGCGCCGCTGCCGCTCTCGTCTACAA
CAACATC
GAGGGCAGCATGGCCGGCACCCTCGGCGGACCTACTTCTGAGCTGGGCCCCTACGCCCCCATTGCCGGCATTTCTCTCG
CCGACG
GCCAGGCCCTCATCCAGATGATTCAGGCCGGCACCGTCACCGCCAACCTCTGGATCGACAGCAAGGTCGAGAACCGCAC
CACCTA
CAACGTCATTGCCCAGACCAAGGGCGGCGACCCCAACAACGTCGTCGCTCTCGGCGGCCACACCGACTCTGTTGAGGCT
GGCCCT
GGCATCAACGACGACGGCAGCGGCATCATCAGCAACCTCGTCGTCGCCAAGGCCCTCACCCGCTTCAGCGTCAAGAACG
CCGTCC
GCTTCTGCTTCTGGACCGCCGAAGAGTTCGGCCTCCTCGGCAGCAGCTACTACGTCAACAGCCTCAACGCCACCGAGAA
GGCCAA
GATCCGCCTCTACCTCAACTTCGACATGATCGCCAGCCCCAACTACGCCCTCATGATCTACGACGGCGACGGCAGCGCC
TTCAACC
TCACTGGCCCTGCTGGCAGCGCCCAGATCGAGCGCCTCTTCGAGGACTACTACAAGAGCATCCGCAAGCCCTTCGTCCC
CACCGA
GTTCAACGGCCGCAGCGACTACGAGGCCTTCATCCTCAACGGCATCCCCGCTGGCGGCATCTTCACTGGCGCCGAGGCC
ATCAAG
ACCGAGGAACAGGCCAAGCTGTTCGGCGGCCAGGCTGGCGTCGCCCTCGATGCCAACTACCACGCCAAGGGCGACAACA
TGACC
AACCTCAACCGCGAGGCCTTCCTCATCAACAGCAAGGCCACCGCCTTCGCCGTCGCCACCTACGCCAACTCCCTCGACA
GCATCC
CCAGCCGCAACATGAGCACCGTCGTCAAGCGCAGCCAGCTTGAGCAGGCCAAGAAGTCCACCCCCCACACCCACACTGG
CGGCA
CCGGCTGCTACAAGGACCGCGTCGAACAGTAAGACCCAGCTTTCTTGTACAAAGTGGTGATCGCGCCAGCTCCGTGCGA
AAGCCT
GACGCACCGGTAGATTCTTGGTGAGCCCGTATCATGACGGCGGCGGGAGCTACATGGCCCCGGGTGATTTATTTTTTTT
GTATCTA
CTTCTGACCCTTTTCAAATATACGGTCAACTCATCTTTCACTGGAGATGCGGCCTGCTTGGTATTGCGATGTTGTCAGC
TTGGCAAA
TTGTGGCTTTCGAAAACACAAAACGATTCCTTAGTAGCCATGCATTTTAAGATAACGGAATAGAAGAAAGAGGAAATTA
AAAAA
AAAAAAAAAACAAACATCCCGTTCATAACCCGTAGAATCGCCGCTCTTCGGCTAGCTAGTTACGCTTGTTTATTTACGA
CAAGATC
TAGAAGATTCGAGATAGAATAATAATAATAACAACAATTTGCCTCTTCTTTCCACCTTTTCAGTCTTACTCTCCCTTCT
GACATTGA
ACGCCTCAATCAGTCAGTCGCCTTGTACTTGGCACGGTAATCCTCCGTGTTCTTGATATCCTCAGGGGTAGCAAAGCCC
TTCATGC
CATCGATAATGTCATCCAGAGTGAGGATGGCAAAGATGGGGATGCCGTACTCCTTCCTCAGCTCGCCAATGGCACTCGG
TCCAGG
CTTGGAGTCGTCGCCATCCGCAGCGGGGAGCTTCTCCATGCGGTCCAGGGCCACGACGATGCCGGCGACGATGCCGCCC
TCCTTG
GTGATCTTCTCAATGGCGTCCCTCTTGGCGGTGCCGGCGGTGATGACGTCGTCGACAATCAGGACCCTCTTGCCCTTGA
GCGAAGC
GCCGACGATGTTGCCGCCCTCGCCGTGGTCCTTGGCCTCCTTGCGGTCAAACGAGTAGGAGACGCGGTCCAGGTTCTGG
GGCGCC
AGCTCGCCGAGCTTGATGGTGATGGCGGAGCACAGCGGGATGCCCTTGTAGGCCGGGCCGAAGACGATGTCGAACTCTA
GGCCG
GCCTTCTCCTGGGCCTCGATGATGGTCTTTGCAAAGGCGGAGGCGATGGCGCCGGCGAGGCGCGCCGTGTGGAATTCGC
CCGCGT
TGAAGAAGTAGGGGGATATCCGCTTGGACTTGAGCTCGAAGCTGCCAAACTTGAGGACGCCGCCGTCGATGGCGGATTT
GAGGA
AGTCCTGCTTGTAGGCAGGCAGCTGGGAGGTGGTAGCCATTCTGTTGGATTTGGATAGTGTCCTTATTCTCTGATTTGA
ACAGTAG
ATCAGGACGAGTGAGAGGGATGCAGAGGTTGGATTGGAGTGGTTGAGCTATAAAATTTAGAGGCGCGCCGTATCGAGTT
TTCACA
TGGAAGTCAAAGCGTACAGTGCGAGCTTGTACGTTGGTCTTAGTATCCCACAAGCTTCTGTCTAGGTATGATGATGGCT
ATAAGTC
ACCCAAGGCAGAACTCATCTTGAAGATTGTCTAGAGTGATTTTACCGCTGATGAAATGACTGGACTCCCTCCTCCTGCT
CTTATAC
GAAAAATTGCCTGACTCTGCAAAGGTTGTTTGTCTTGGAAGATGATGTGCCC CCCCATCGCTCTTATCTCATAC
CCCGCCATCTTTC
TAGATTCTCATCTTCAACAAGAGGGGCAATCCATGATCTGCGATCCAGATGTGCTTCTGGCCTCATACTCTGCCTTCAG
GTTGATG
TTCACTTAATTGGTGACGAATTCAGCTGATTTGCTGCAGTATGCTTTGTGTTGGTTCTTTCCAGGCTTGTGCCAGCCAT
GAGCGCTT
TGAGAGCATGTTGTCACCTATAAACTCGAGTAACGGCCACATATTGTTCACTACTTGAATCACATACCTAATTTTGATA
GAATTGA
CATGTTTAAAGAGCTGAGGTAGCTTTAATGCCTCTGAAGTATTGTGACACAGCTTCTCACAGAGTGAGAATGAAAAGTT
GGACTC
CCC CTAATGAAGTAAAAGTTTCGTCTCTGAACGGTGAAGAGCATAGATCC
GGCATCAACTACCTGGCTAGACTACGACGTCAATT
CTGC GGCCTTTTGAC
CTTTATATATGTCCATTAATGCAATAGATTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCCC
AATTTCGCAGATCAAAGTGGAC GTTATAGCATCATAACTAAGCTCAGTTGCTGAGGGAAGCC GTCTACTAC
CTTAGCCCATCCATC
CAGCTCCATACCTTGATACTTTAGACGTGAAGCAATTCACACTGTACGTCTCGCAGCTCTCCTTCCCGCTCTTGCTTCC
CCACTGGG
GTC CATGGTGCGTGTATCGTCC CCTC
CTTAATTAAGGCCATTTAGGCCGTTGCTGGCGTTTTTCCATAGGCTCCGCC CCCCTGACGA
GCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAAC CCGACAGGACTATAAAGATAC CAGGCGTTTCC
CCCTGGAAGCTC
CCTCGTGCGCTCTCCTGTTCCGACC CTGCC GCTTACCGGATACCTGTCCGC CTTTCTCCCTTCGGGAAGC
GTGGCGC TTT CT CATAG
CT CACGCT GTAGGTATCT CAGTT CGGTGTAGGTCGTTCGC TCC AAGCT GGGCTGT GTGCAC GAACC
CCCC GTTCAGCCCGACCGCT
GCGCCTTATCCGGTAACTATCGTCTTGAGTC CAACCC GGTAAGACACGACTTATC GCCACTGGCAGCAGC
CACTGGTAACAGGAT
TAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTA
TTTGGT
ATCTGCGCTCTGCTGAAGCCAGTTAC CTTCGGAAAAAGAGTTGGTAGCTCTTGATCC GGCAAACAAAC
CACCGCTGGTAGC GGTG
GTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTC
TGACGCT
CAGTGGAACGAAAACTCACGTTAAGGC CTGCAGGGCCGATTTTGGTCATGAGATTATCAAAAAGGATCTTCAC
CTAGATCCTTTT
AAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGT
GAGGCA
CCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGG
GCTTACC
ATCTGGCC CCAGTGCTGCAATGATAC CGCGAGACCCACGCTCAC
CGGCTCCAGATTTATCAGCAATAAACCAGCCAGCC GGAAGG
GCCGAGC GCAGAAGTGGTCCTGCAACTTTATCC GCCTCCATCCAGTCTATTAATTGTTGCC
GGGAAGCTAGAGTAAGTAGTTCGCC
AGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTC
AGCTCCGG
TT CCCAAC GATCAAGGC GAGTTACATGATCCCC CAT GTTGT GCAAAAAAGCGGTTAGCTC CTTC
GGTCCTCCGATCGTTGTCAGAA
GTAAGTTGGC CGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGC
CATCCGTAAGATGCTTTTCTG
TGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGAC CGAGTTGCTCTTGC
CCGGCGTCAATACGGGATAA
TACC GCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTC
GGGGCGAAAACTCTCAAGGATCTTACCGCTG
TT GAGATCC AGTT CGATGTAAC CCACTC
GTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAA
AACAGGAAGGCAAAATGC CGCAAAAAAGGGAATAAGGGCGACAC
GGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTAT
TGAAGCATTTATCAGGGTTATTGTCTCATGGCCATTTAGGCCT (Seq ID no 9)
CTAGAGTTGTGAAGTCGGTAATCCCGCTGTATAGTAATACGAGTCGCATCTAAATACTCCGAAGCTGCTGCGAACCCGG
AGAATC
GAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAA
TCATGG
CGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTCCCGAAAAAACTCGGAGATTCCTAAGT
AGCGAT
GGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAAC
TGTTCC
GTACCCCACCTCTTCTCAACCTTTGGCGTTTCCCTGATTCAGCGTACCCGTACAAGTCGTAATCACTATTAACCCAGAC
TGACCGG
ACGTGTTTTGCCCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGACCGACTGGGGCTGTTCGA
AGCCCGA
ATGTAGGATTGTTATCCGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGCCTCGAAGGTTCA
CGGCAA
GGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCCGCAATGCAGCATCACTGGAAAATACAAACCAATGGC
TAAAA
GTACATAAGTTAATGCCTAAAGAAGTCATATACCAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATT
TGCCAA
CGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGG
TGATCCC
CCAATTGGGTCGCTTGTTTGTTCCGGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTCGGAGCGTTTTGCATAC
AACCAA
GGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAG
GTTTGT
CTGCCGATACGACGAATACTGTATAGTCACTTCTGATGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCA
AAAGAT
TGAGTTGAAACTGCCTAAGATCTCGGGCCCTCGGGCCTTCGGCCTTTGGGTGTACATGTTTGTGCTCCGGGCAAATGCA
AAGTGTG
GTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGT
TTCGAA
TAGAAAGAGAAGCTTAGCCAAGAACAATAGCCGATAAAGATAGCCTCATTAAACGGAATGAGCTAGTAGGCAAAGTCAG
CGAAT
GTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGAC
TTGTACA
CCATCTTTTGAGGCACAGAAACCCAATAGTCAACCATCACAAGTTTGTACAAAAAAGCAGGCTTCACCATGCAGACCTT
CGGTGC
TTTTCTCGTTTCCTTCCTCGCCGCCAGGTAAGTTGGCCTTGATGAACCATATCATATATCGCCGAGAAGTGGACCGCGT
GCTGAGA
CTGAGACAGCGGCCTGGCCGCGGCCAACGGACCTGGATGGGATTGGAAGCCCCCCGTCCACCCCAAGGTCCTCCCCCAG
ATGATC
CACCTCTGGGACCTCATGCACGGCGCCCAGAAGCTCGAAGATTTCGCCTACGCCTACCCCGAGCGCAACCGCGTCTTTG
GCGGCC
CTGCCCACGAGGACACCGTCAACTACCTCTACCGCGAGCTGAAGAAGACCGGCTACTACGACGTCTACAAGCAGCCCCA
GGTCCA
CCAGTGGACCCGAGCCGATCAGGCCCTCACCGTCGACGGCAAGAGCTACGTCGCCACCACCATGACCTACAGCCCCAGC
GTCAAC
GTCACCGCCCCTCTCGCCGTCGTCAACAACCTCGGCTGCGTCGAGAGCGACTACCCCGCCGACCTCAAGGGCAAGATCG
CCCTCG
TTTCTCGCGGCGAGTGCCCCTTCGCCACCAAGTCTGTCCTCAGCGCCAAGGCTGGCGCCGCTGCCGCTCTCGTCTACAA
CAACATC
GAGGGCAGCATGGCCGGCACCCTCGGCGGACCTACTTCTGAGCTGGGCCCCTACGCCCCCATTGCCGGCATTTCTCTCG
CCGACG
GCCAGGCCCTCATCCAGATGATTCAGGCCGGCACCGTCACCGCCAACCTCTGGATCGACAGCAAGGTCGAGAACCGCAC
CACCTA
CAACGTCATTGCCCAGACCAAGGGCGGCGACCCCAACAACGTCGTCGCTCTCGGCGGCCACACCGACTCTGTTGAGGCT
GGCCCT
GGCATCAACGACGACGGCAGCGGCATCATCAGCAACCTCGTCGTCGCCAAGGCCCTCACCCGCTTCAGCGTCAAGAACG
CCGTCC
GCTTCTGCTTCTGGACCGCCGAAGAGTTCGGCCTCCTCGGCAGCAGCTACTACGTCAACAGCCTCAACGCCACCGAGAA
GGCCAA
GATCCGCCTCTACCTCAACTTCGACATGATCGCCAGCCCCAACTACGCCCTCATGATCTACGACGGCGACGGCAGCGCC
TTCAACC
TCACTGGCCCTGCTGGCAGCGCCCAGATCGAGCGCCTCTTCGAGGACTACTACAAGAGCATCCGCAAGCCCTTCGTCCC
CACCGA
GTTCAACGGCCGCAGCGACTACGAGGCCTTCATCCTCAACGGCATCCCCGCTGGCGGCATCTTCACTGGCGCCGAGGCC
ATCAAG
ACCGAGGAACAGGCCAAGCTGTTCGGCGGCCAGGCTGGCGTCGCCCTCGATGCCAACTACCACGCCAAGGGCGACAACA
TGACC
AACCTCAACCGCGAGGCCTTCCTCATCAACAGCAAGGCCACCGCCTTCGCCGTCGCCACCTACGCCAACTCCCTCGACA
GCATCC
CCAGCCGCAACATGAGCACCGTCGTCAAGCGCAGCCAGCTTGAGCAGGCCAAGAAGTCCACCCCCCACACCCACACTGG
CGGCA
CCGGCTGCTACAAGGACCGCGTCGAACAGTAAGACCCAGCTTTCTTGTACAAAGTGGTGATCGCGCCAGCTCCGTGCGA
AAGCCT
GACGCACCGGTAGATTCTTGGTGAGCCCGTATCATGACGGCGGCGGGAGCTACATGGCCCCGGGTGATTTATTTTTTTT
GTATCTA
CTTCTGACCCTTTTCAAATATACGGTCAACTCATCTTTCACTGGAGATGCGGCCTGCTTGGTATTGCGATGTTGTCAGC
TTGGCAAA
TTGTGGCTTTCGAAAACACAAAACGATTCCTTAGTAGCCATGCATTTTAAGATAACGGAATAGAAGAAAGAGGAAATTA
AAAAA
AAAAAAAAAACAAACATCCCGTTCATAACCCGTAGAATCGCCGCTCTTCGGCTAGCTAGTTACGCTTGTTTATTTACGA
CAAGATC
TAGAAGATTCGAGATAGAATAATAATAATAACAACAATTTGCCTCTTCTTTCCACCTTTTCAGTCTTACTCTCCCTTCT
GACATTGA
ACGCCTCAATCAGTCAGTCGCCTTGTACTTGGCACGGTAATCCTCCGTGTTCTTGATATCCTCAGGGGTAGCAAAGCCC
TTCATGC
CATCGATAATGTCATCCAGAGTGAGGATGGCAAAGATGGGGATGCCGTACTCCTTCCTCAGCTCGCCAATGGCACTCGG
TCCAGG
CTTGGAGTCGTCGCCATCCGCAGCGGGGAGCTTCTCCATGCGGTCCAGGGCCACGACGATGCCGGCGACGATGCCGCCC
TCCTTG
GTGATCTTCTCAATGGCGTCCCTCTTGGCGGTGCCGGCGGTGATGACGTCGTCGACAATCAGGACCCTCTTGCCCTTGA
GCGAAGC
GCCGACGATGTTGCCGCCCTCGCCGTGGTCCTTGGCCTCCTTGCGGTCAAACGAGTAGGAGACGCGGTCCAGGTTCTGG
GGCGCC
AGCTCGCCGAGCTTGATGGTGATGGCGGAGCACAGCGGGATGCCCTTGTAGGCCGGGCCGAAGACGATGTCGAACTCTA
GGCCG
GCCTTCTCCTGGGCCTCGATGATGGTCTTTGCAAAGGCGGAGGCGATGGCGCCGGCGAGGCGCGCCGTGTGGAATTCGC
CCGCGT
TGAAGAAGTAGGGGGATATCCGCTTGGACTTGAGCTCGAAGCTGCCAAACTTGAGGACGCCGCCGTCGATGGCGGATTT
GAGGA
AGTCCTGCTTGTAGGCAGGCAGCTGGGAGGTGGTAGCCATTCTGTTGGATTTGGATAGTGTCCTTATTCTCTGATTTGA
ACAGTAG
ATCAGGACGAGTGAGAGGGATGCAGAGGTTGGATTGGAGTGGTTGAGCTATAAAATTTAGAGGCGCGCCGTATCGAGTT
TTCACA
TGGAAGTCAAAGCGTACAGTGCGAGCTTGTACGTTGGTCTTAGTATCCCACAAGCTTCTGTCTAGGTATGATGATGGCT
ATAAGTC
ACCCAAGGCAGAACTCATCTTGAAGATTGTCTAGAGTGATTTTACCGCTGATGAAATGACTGGACTCCCTCCTCCTGCT
CTTATAC
GAAAAATTGCCTGACTCTGCAAAGGTTGTTTGTCTTGGAAGATGATGTGCCC CCCCATCGCTCTTATCTCATAC
CCCGCCATCTTTC
TAGATTCTCATCTTCAACAAGAGGGGCAATCCATGATCTGCGATCCAGATGTGCTTCTGGCCTCATACTCTGCCTTCAG
GTTGATG
TTCACTTAATTGGTGACGAATTCAGCTGATTTGCTGCAGTATGCTTTGTGTTGGTTCTTTCCAGGCTTGTGCCAGCCAT
GAGCGCTT
TGAGAGCATGTTGTCACCTATAAACTCGAGTAACGGCCACATATTGTTCACTACTTGAATCACATACCTAATTTTGATA
GAATTGA
CATGTTTAAAGAGCTGAGGTAGCTTTAATGCCTCTGAAGTATTGTGACACAGCTTCTCACAGAGTGAGAATGAAAAGTT
GGACTC
CCC CTAATGAAGTAAAAGTTTCGTCTCTGAACGGTGAAGAGCATAGATCC
GGCATCAACTACCTGGCTAGACTACGACGTCAATT
CTGC GGCCTTTTGAC
CTTTATATATGTCCATTAATGCAATAGATTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCCC
AATTTCGCAGATCAAAGTGGAC GTTATAGCATCATAACTAAGCTCAGTTGCTGAGGGAAGCC GTCTACTAC
CTTAGCCCATCCATC
CAGCTCCATACCTTGATACTTTAGACGTGAAGCAATTCACACTGTACGTCTCGCAGCTCTCCTTCCCGCTCTTGCTTCC
CCACTGGG
GTC CATGGTGCGTGTATCGTCC CCTC
CTTAATTAAGGCCATTTAGGCCGTTGCTGGCGTTTTTCCATAGGCTCCGCC CCCCTGACGA
GCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAAC CCGACAGGACTATAAAGATAC CAGGCGTTTCC
CCCTGGAAGCTC
CCTCGTGCGCTCTCCTGTTCCGACC CTGCC GCTTACCGGATACCTGTCCGC CTTTCTCCCTTCGGGAAGC
GTGGCGC TTT CT CATAG
CT CACGCT GTAGGTATCT CAGTT CGGTGTAGGTCGTTCGC TCC AAGCT GGGCTGT GTGCAC GAACC
CCCC GTTCAGCCCGACCGCT
GCGCCTTATCCGGTAACTATCGTCTTGAGTC CAACCC GGTAAGACACGACTTATC GCCACTGGCAGCAGC
CACTGGTAACAGGAT
TAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTA
TTTGGT
ATCTGCGCTCTGCTGAAGCCAGTTAC CTTCGGAAAAAGAGTTGGTAGCTCTTGATCC GGCAAACAAAC
CACCGCTGGTAGC GGTG
GTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTC
TGACGCT
CAGTGGAACGAAAACTCACGTTAAGGC CTGCAGGGCCGATTTTGGTCATGAGATTATCAAAAAGGATCTTCAC
CTAGATCCTTTT
AAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGT
GAGGCA
CCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGG
GCTTACC
ATCTGGCC CCAGTGCTGCAATGATAC CGCGAGACCCACGCTCAC
CGGCTCCAGATTTATCAGCAATAAACCAGCCAGCC GGAAGG
GCCGAGC GCAGAAGTGGTCCTGCAACTTTATCC GCCTCCATCCAGTCTATTAATTGTTGCC
GGGAAGCTAGAGTAAGTAGTTCGCC
AGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTC
AGCTCCGG
TT CCCAAC GATCAAGGC GAGTTACATGATCCCC CAT GTTGT GCAAAAAAGCGGTTAGCTC CTTC
GGTCCTCCGATCGTTGTCAGAA
GTAAGTTGGC CGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGC
CATCCGTAAGATGCTTTTCTG
TGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGAC CGAGTTGCTCTTGC
CCGGCGTCAATACGGGATAA
TACC GCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTC
GGGGCGAAAACTCTCAAGGATCTTACCGCTG
TT GAGATCC AGTT CGATGTAAC CCACTC
GTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAA
AACAGGAAGGCAAAATGC CGCAAAAAAGGGAATAAGGGCGACAC
GGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTAT
TGAAGCATTTATCAGGGTTATTGTCTCATGGCCATTTAGGCCT (Seq ID no 9)
[00293] SEQ ID NO: 10 = Nucleotide sequence of the pTrex8gM TR1032 expression construct.
CTAGAGTTGTGAAGTC GGTAATC CCGCTGTATAGTAATAC GAGTCGCATCTAAATACTCC
GAAGCTGCTGCGAACCCGGAGAATC
GAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAA
TCATGG
CGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTC CCGAAAAAACTC GGAGATTC
CTAAGTAGCGAT
GGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAAC
TGTTCC
GTAC CCCACCTCTTCTCAACCTTTGGC GTTT CC CTGATTCAGC GTACC
CGTACAAGTCGTAATCACTATTAACCCAGACTGACC GG
ACGTGTTTTGC CCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGAC
CGACTGGGGCTGTTC GAAGCCCGA
ATGTAGGATTGTTATC CGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGC
CTCGAAGGTTCACGGCAA
GGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCC GCAATGCAGCATCACTGGAAAATACAAAC
CAATGGCTAAAA
GTACATAAGTTAATGC CTAAAGAAGTCATATAC
CAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAA
CGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGG
TGATCCC
CCAATTGGGTCGCTTGTTTGTTCCGGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTCGGAGCGTTTTGCATAC
AACCAA
GGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAG
GTTTGT
CTGCCGATACGACGAATACTGTATAGTCACTTCTGATGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCA
AAAGAT
TGAGTTGAAACTGCCTAAGATCTCGGGCCCTCGGGCCTTCGGCCTTTGGGTGTACATGTTTGTGCTCCGGGCAAATGCA
AAGTGTG
GTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGT
TTCGAA
TAGAAAGAGAAGCTTAGCCAAGAACAATAGCCGATAAAGATAGCCTCATTAAACGGAATGAGCTAGTAGGCAAAGTCAG
CGAAT
GTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGAC
TTGTACA
CCATCTTTTGAGGCACAGAAACCCAATAGTCAACCATCACAAGTTTGTACAAAAAAGCAGGCTTCACCATGCAGACCTT
CGGTGC
TTTTCTCGTTTCCTTCCTCGCCGCCAGGTAAGTTGGCCTTGATGAACCATATCATATATCGCCGAGAAGTGGACCGCGT
GCTGAGA
CTGAGACAGCGGCCTGGCCGCGGCCGGTGGACATGGTGGATCTTCAGGCCTCGGCTGCGACAGCCAGCGCCCTCTTGTC
AGCAGC
GAGAAGCTCCAGAGCCTGATCAAGAAGGAAGATCTCCTCGCCGGCAGCCAAGAGCTTCAGGACATTGCCACTGCCCACG
GCGGC
CACCGAGCCTTTGGAAGCTCTGGCCACAACGCCACCGTCGACTTTCTCTACTACACCCTCAAGGCCCTCGACTACTACA
ACGTCAC
CAAGCAGCCCTTCAAGGAAATCTTCAGCAGCGGCACCGGCAGCCTCACCGTGGACGGCGAGGACATCGAGGCCGAGACT
CTCAC
CTACACCCCCAGCGGCAGCGCCACCGACAAGCCTGTCGTCGTCGTCGCCAACGTCGGCTGCGACGCCGCCGATTACCCT
GCTGAG
GTCGCCGGCAACATTGCCCTCATCAAGCGCGGCACGTGCACCTTCAGCCAGAAGTCCGTCAACGCCAAGGCCGCTGGCG
CCGTCG
CCGCCATCATCTACAACAACGCCGAGGGCAAGCTCAGCGGAACCCTCGGCCAGCCCTTCCTCGACTACGCTCCCGTCCT
CGGCAT
CACCCTTGAGGCCGGCGAGGCCCTCCTCGCCAAGCTCGCTGGTGGCCCTGTCACCGCCACCCTCCAGATTGACGCCCTC
GTCGAG
GAACGCGTCACCTACAACGTCATTGCCGAGACTAAGGAAGGCGACCACAGCAACGTCCTCGTCCTCGGCGGCCACACCG
ATAGC
GTCCCTGCTGGCCCTGGCATCAACGACGACGGCAGCGGCACCATCGGCATGCTCACTGTCGCCAAGGCCCTCACCAAGT
TCCGCG
TCAAGAACGCCGTCCGCTTCGCCTTCTGGTCCGCCGAGGAATACGGCCTCCTCGGCAGCTACGCCTACATCAAGAGCAT
CAACAG
CTCTGCCGCCGAGCTGAGCAAGATCCGCGCCTACCTCAACTTCGACATGATCGCCAGCCCCAACTACATCTACGGCATC
TACGAC
GGCGACGGCAACGCCTTCAACCTCACTGGCCCTGCCGGCAGCGACGTCATCGAGCGCAACTTCGAGAACTTCTTCAAGC
GCAAGC
ACACCCCCTCCGTCCCCACCGAGTTTAGCGGCCGATCTGACTACGCCGCCTTCATCGAGAACGGCATCCCCAGCGGCGG
ACTCTTC
ACTGGCGCCGAGGTCCTCAAGACCGAGCGCGAGGCTGAGCTGTTTGGCGGCCGAGCTGGCGTCGCCTACGACGTCAACT
ACCACC
AGGCCGGCGACACCGTCGACAACCTCGCCCTCGACGCCTTCCTGCTCAACACCAAGGCCATTGCCGACAGCGTCGCCAC
CTACGC
CCTCAGCTTTGACGGCCTCCCTCGCGTCGACGGCAAGAAGCGACGTTGGGACGCTCACCGAGCCCGCATGCTCAAGCGA
TCTGCT
GGCTCTCACGGCCACGCCCACCTTCACTCTGGCCCTTGTGGCGGCGGAGCCAGCATCTAAGACCCAGCTTTCTTGTACA
AAGTGGT
GATCGCGCCAGCTCCGTGCGAAAGCCTGACGCACCGGTAGATTCTTGGTGAGCCCGTATCATGACGGCGGCGGGAGCTA
CATGGC
CCCGGGTGATTTATTTTTTTTGTATCTACTTCTGACCCTTTTCAAATATACGGTCAACTCATCTTTCACTGGAGATGCG
GCCTGCTTG
GTATTGCGATGTTGTCAGCTTGGCAAATTGTGGCTTTCGAAAACACAAAACGATTCCTTAGTAGCCATGCATTTTAAGA
TAACGGA
ATAGAAGAAAGAGGAAATTAAAAAAAAAAAAAAAACAAACATCCCGTTCATAACCCGTAGAATCGCCGCTCTTCGGCTA
GCTAG
TTACGCTTGTTTATTTACGACAAGATCTAGAAGATTCGAGATAGAATAATAATAATAACAACAATTTGCCTCTTCTTTC
CACCTTTT
CAGTCTTACTCTCCCTTCTGACATTGAACGCCTCAATCAGTCAGTCGCCTTGTACTTGGCACGGTAATCCTCCGTGTTC
TTGATATC
CTCAGGGGTAGCAAAGCCCTTCATGCCATCGATAATGTCATCCAGAGTGAGGATGGCAAAGATGGGGATGCCGTACTCC
TTCCTC
AGCTCGCCAATGGCACTCGGTCCAGGCTTGGAGTCGTCGCCATCCGCAGCGGGGAGCTTCTCCATGCGGTCCAGGGCCA
CGACGA
TGCCGGCGACGATGCCGCCCTCCTTGGTGATCTTCTCAATGGCGTCCCTCTTGGCGGTGCCGGCGGTGATGACGTCGTC
GACAATC
AGGACCCTCTTGCCCTTGAGCGAAGCGCCGACGATGTTGCCGCCCTCGCCGTGGTCCTTGGCCTCCTTGCGGTCAAACG
AGTAGG
AGACGCGGTCCAGGTTCTGGGGCGCCAGCTCGCCGAGCTTGATGGTGATGGCGGAGCACAGCGGGATGCCCTTGTAGGC
CGGGC
CGAAGACGATGTCGAACTCTAGGCCGGCCTTCTCCTGGGCCTCGATGATGGTCTTTGCAAAGGCGGAGGCGATGGCGCC
GGCGAG
GCGCGCCGTGTGGAATTCGCCCGCGTTGAAGAAGTAGGGGGATATCCGCTTGGACTTGAGCTCGAAGCTGCCAAACTTG
AGGACG
CCGCCGTCGATGGCGGATTTGAGGAAGTCCTGCTTGTAGGCAGGCAGCTGGGAGGTGGTAGCCATTCTGTTGGATTTGG
ATAGTG
TCCTTATTCTCTGATTTGAACAGTAGATCAGGACGAGTGAGAGGGATGCAGAGGTTGGATTGGAGTGGTTGAGCTATAA
AATTTA
GAGGCGCGCCGTATCGAGTTTTCACATGGAAGTCAAAGCGTACAGTGCGAGCTTGTACGTTGGTCTTAGTATCCCACAA
GCTTCTG
TCTAGGTATGATGATGGCTATAAGTCACCCAAGGCAGAACTCATCTTGAAGATTGTCTAGAGTGATTTTACCGCTGATG
AAATGAC
TGGACTCCCTCCTCCTGCTCTTATACGAAAAATTGCCTGACTCTGCAAAGGTTGTTTGTCTTGGAAGATGATGTGCCCC
CCCATCGC
TCTTATCTCATACCCCGCCATCTTTCTAGATTCTCATCTTCAACAAGAGGGGCAATCCATGATCTGCGATCCAGATGTG
CTTCTGGC
CTCATACTCTGCCTTCAGGTTGATGTTCACTTAATTGGTGACGAATTCAGCTGATTTGCTGCAGTATGCTTTGTGTTGG
TTCTTTCC
AGGCTTGTGC CAGCCATGAGCGCTTTGAGAGCATGTTGTCAC CTATAAACTC GAGTAAC
GGCCACATATTGTTCACTACTTGAATC
ACATACCTAATTTTGATAGAATTGACATGTTTAAAGAGCTGAGGTAGCTTTAATGCCTCTGAAGTATTGTGACACAGCT
TCTCACA
GAGTGAGAATGAAAAGTTGGACTCCCCCTAATGAAGTAAAAGTTTCGTCTCTGAACGGTGAAGAGCATAGATCCGGCAT
CAACTA
CCTGGCTAGACTACGACGTCAATTCTGC GGCCTTTTGAC
CTTTATATATGTCCATTAATGCAATAGATTCTTTTTTTTTTTTTTTTTT
TTTTTTTTTTTTTTTTTTTTTTTGCCCAATTTC GCAGATCAAAGTGGAC
GTTATAGCATCATAACTAAGCTCAGTTGCTGAGGGAAG
CCGTCTACTAC CTTAGC CCATC CAT CCAGCTC CATAC
CTTGATACTTTAGACGTGAAGCAATTCACACTGTAC GTCTCGCAGCTCTC
CTTCCCGCTCTTGCTTCC CCACTGGGGTCCATGGTGCGTGTATC GTCC CCTC
CTTAATTAAGGCCATTTAGGCCGTTGCTGGCGTTT
TT CCATAGGC TC CGCCCC
CCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGA
TAC CAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGAC CCTGC CGCTTACCGGATACCTGTC
CGCCTTTCTC CCT
TCGGGAAGCGT GGCGCTTTCT CATAGC TCACGC TGTAGGTAT CT CAGTTC GGTGTAGGTC GTTC
GCTCCAAGCTGGGCTGTGTGCA
CGAAC CCCCC GTTCAGCCC GACC GCTGCGC CTTATCCGGTAACTATCGTCTTGAGTCCAACCC
GGTAAGACACGACTTATCGC CAC
TGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAA
CTACGG
CTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGA
TCCGGCA
AACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGA
TCCTTT
GATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGCCTGCAGGGCCGATTTTGGTCATGAGAT
TATCAA
AAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTC
TGACAGT
TAC CAATGC TTAAT CAGTGAGGCACC TATC TCAGCGAT CT GTCTATTTCGTTCAT CCATAGTTGCC
TGAC TCCC CGTCGTGTAGATA
ACTACGATACGGGAGGGCTTACCATCTGGC CCCAGTGCTGCAATGATACC GCGAGAC CCACGCTCACC GGCTCC
AGATTTATC AG
CAATAAACCAGC CAGCCGGAAGGGC CGAGCGCAGAAGTGGTCCTGCAACTTTATCC
GCCTCCATCCAGTCTATTAATTGTTGC CG
GGAAGCTAGAGTAAGTAGTTC GCCAGTTAATAGTTTGCGCAAC
GTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGT
TT GGTAT GGCTT CATT CAGCTC CGGTTCCCAAC GATCAAGGCGAGTTACATGATCC CCCAT
GTTGTGCAAAAAA GCGGTTAGCT CC
TT CGGTC CTCC
GATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTC
ATGCCATCC GTAAGATGCTTTTCTGTGACTGGTGAGTACTCAAC CAAGTCATTCTGAGAATAGTGTATGC
GGCGACC GAGTTGCTC
TT GCCCGGCGTCAATACGGGATAATACC
GCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGC GA
AAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTT
TTACTTTC
ACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAA
TACTC
ATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGGCCATTTAGGCCT (Seq ID no 10)
CTAGAGTTGTGAAGTC GGTAATC CCGCTGTATAGTAATAC GAGTCGCATCTAAATACTCC
GAAGCTGCTGCGAACCCGGAGAATC
GAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAA
TCATGG
CGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTC CCGAAAAAACTC GGAGATTC
CTAAGTAGCGAT
GGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAAC
TGTTCC
GTAC CCCACCTCTTCTCAACCTTTGGC GTTT CC CTGATTCAGC GTACC
CGTACAAGTCGTAATCACTATTAACCCAGACTGACC GG
ACGTGTTTTGC CCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGAC
CGACTGGGGCTGTTC GAAGCCCGA
ATGTAGGATTGTTATC CGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGC
CTCGAAGGTTCACGGCAA
GGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCC GCAATGCAGCATCACTGGAAAATACAAAC
CAATGGCTAAAA
GTACATAAGTTAATGC CTAAAGAAGTCATATAC
CAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAA
CGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGG
TGATCCC
CCAATTGGGTCGCTTGTTTGTTCCGGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTCGGAGCGTTTTGCATAC
AACCAA
GGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAG
GTTTGT
CTGCCGATACGACGAATACTGTATAGTCACTTCTGATGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCA
AAAGAT
TGAGTTGAAACTGCCTAAGATCTCGGGCCCTCGGGCCTTCGGCCTTTGGGTGTACATGTTTGTGCTCCGGGCAAATGCA
AAGTGTG
GTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGT
TTCGAA
TAGAAAGAGAAGCTTAGCCAAGAACAATAGCCGATAAAGATAGCCTCATTAAACGGAATGAGCTAGTAGGCAAAGTCAG
CGAAT
GTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGAC
TTGTACA
CCATCTTTTGAGGCACAGAAACCCAATAGTCAACCATCACAAGTTTGTACAAAAAAGCAGGCTTCACCATGCAGACCTT
CGGTGC
TTTTCTCGTTTCCTTCCTCGCCGCCAGGTAAGTTGGCCTTGATGAACCATATCATATATCGCCGAGAAGTGGACCGCGT
GCTGAGA
CTGAGACAGCGGCCTGGCCGCGGCCGGTGGACATGGTGGATCTTCAGGCCTCGGCTGCGACAGCCAGCGCCCTCTTGTC
AGCAGC
GAGAAGCTCCAGAGCCTGATCAAGAAGGAAGATCTCCTCGCCGGCAGCCAAGAGCTTCAGGACATTGCCACTGCCCACG
GCGGC
CACCGAGCCTTTGGAAGCTCTGGCCACAACGCCACCGTCGACTTTCTCTACTACACCCTCAAGGCCCTCGACTACTACA
ACGTCAC
CAAGCAGCCCTTCAAGGAAATCTTCAGCAGCGGCACCGGCAGCCTCACCGTGGACGGCGAGGACATCGAGGCCGAGACT
CTCAC
CTACACCCCCAGCGGCAGCGCCACCGACAAGCCTGTCGTCGTCGTCGCCAACGTCGGCTGCGACGCCGCCGATTACCCT
GCTGAG
GTCGCCGGCAACATTGCCCTCATCAAGCGCGGCACGTGCACCTTCAGCCAGAAGTCCGTCAACGCCAAGGCCGCTGGCG
CCGTCG
CCGCCATCATCTACAACAACGCCGAGGGCAAGCTCAGCGGAACCCTCGGCCAGCCCTTCCTCGACTACGCTCCCGTCCT
CGGCAT
CACCCTTGAGGCCGGCGAGGCCCTCCTCGCCAAGCTCGCTGGTGGCCCTGTCACCGCCACCCTCCAGATTGACGCCCTC
GTCGAG
GAACGCGTCACCTACAACGTCATTGCCGAGACTAAGGAAGGCGACCACAGCAACGTCCTCGTCCTCGGCGGCCACACCG
ATAGC
GTCCCTGCTGGCCCTGGCATCAACGACGACGGCAGCGGCACCATCGGCATGCTCACTGTCGCCAAGGCCCTCACCAAGT
TCCGCG
TCAAGAACGCCGTCCGCTTCGCCTTCTGGTCCGCCGAGGAATACGGCCTCCTCGGCAGCTACGCCTACATCAAGAGCAT
CAACAG
CTCTGCCGCCGAGCTGAGCAAGATCCGCGCCTACCTCAACTTCGACATGATCGCCAGCCCCAACTACATCTACGGCATC
TACGAC
GGCGACGGCAACGCCTTCAACCTCACTGGCCCTGCCGGCAGCGACGTCATCGAGCGCAACTTCGAGAACTTCTTCAAGC
GCAAGC
ACACCCCCTCCGTCCCCACCGAGTTTAGCGGCCGATCTGACTACGCCGCCTTCATCGAGAACGGCATCCCCAGCGGCGG
ACTCTTC
ACTGGCGCCGAGGTCCTCAAGACCGAGCGCGAGGCTGAGCTGTTTGGCGGCCGAGCTGGCGTCGCCTACGACGTCAACT
ACCACC
AGGCCGGCGACACCGTCGACAACCTCGCCCTCGACGCCTTCCTGCTCAACACCAAGGCCATTGCCGACAGCGTCGCCAC
CTACGC
CCTCAGCTTTGACGGCCTCCCTCGCGTCGACGGCAAGAAGCGACGTTGGGACGCTCACCGAGCCCGCATGCTCAAGCGA
TCTGCT
GGCTCTCACGGCCACGCCCACCTTCACTCTGGCCCTTGTGGCGGCGGAGCCAGCATCTAAGACCCAGCTTTCTTGTACA
AAGTGGT
GATCGCGCCAGCTCCGTGCGAAAGCCTGACGCACCGGTAGATTCTTGGTGAGCCCGTATCATGACGGCGGCGGGAGCTA
CATGGC
CCCGGGTGATTTATTTTTTTTGTATCTACTTCTGACCCTTTTCAAATATACGGTCAACTCATCTTTCACTGGAGATGCG
GCCTGCTTG
GTATTGCGATGTTGTCAGCTTGGCAAATTGTGGCTTTCGAAAACACAAAACGATTCCTTAGTAGCCATGCATTTTAAGA
TAACGGA
ATAGAAGAAAGAGGAAATTAAAAAAAAAAAAAAAACAAACATCCCGTTCATAACCCGTAGAATCGCCGCTCTTCGGCTA
GCTAG
TTACGCTTGTTTATTTACGACAAGATCTAGAAGATTCGAGATAGAATAATAATAATAACAACAATTTGCCTCTTCTTTC
CACCTTTT
CAGTCTTACTCTCCCTTCTGACATTGAACGCCTCAATCAGTCAGTCGCCTTGTACTTGGCACGGTAATCCTCCGTGTTC
TTGATATC
CTCAGGGGTAGCAAAGCCCTTCATGCCATCGATAATGTCATCCAGAGTGAGGATGGCAAAGATGGGGATGCCGTACTCC
TTCCTC
AGCTCGCCAATGGCACTCGGTCCAGGCTTGGAGTCGTCGCCATCCGCAGCGGGGAGCTTCTCCATGCGGTCCAGGGCCA
CGACGA
TGCCGGCGACGATGCCGCCCTCCTTGGTGATCTTCTCAATGGCGTCCCTCTTGGCGGTGCCGGCGGTGATGACGTCGTC
GACAATC
AGGACCCTCTTGCCCTTGAGCGAAGCGCCGACGATGTTGCCGCCCTCGCCGTGGTCCTTGGCCTCCTTGCGGTCAAACG
AGTAGG
AGACGCGGTCCAGGTTCTGGGGCGCCAGCTCGCCGAGCTTGATGGTGATGGCGGAGCACAGCGGGATGCCCTTGTAGGC
CGGGC
CGAAGACGATGTCGAACTCTAGGCCGGCCTTCTCCTGGGCCTCGATGATGGTCTTTGCAAAGGCGGAGGCGATGGCGCC
GGCGAG
GCGCGCCGTGTGGAATTCGCCCGCGTTGAAGAAGTAGGGGGATATCCGCTTGGACTTGAGCTCGAAGCTGCCAAACTTG
AGGACG
CCGCCGTCGATGGCGGATTTGAGGAAGTCCTGCTTGTAGGCAGGCAGCTGGGAGGTGGTAGCCATTCTGTTGGATTTGG
ATAGTG
TCCTTATTCTCTGATTTGAACAGTAGATCAGGACGAGTGAGAGGGATGCAGAGGTTGGATTGGAGTGGTTGAGCTATAA
AATTTA
GAGGCGCGCCGTATCGAGTTTTCACATGGAAGTCAAAGCGTACAGTGCGAGCTTGTACGTTGGTCTTAGTATCCCACAA
GCTTCTG
TCTAGGTATGATGATGGCTATAAGTCACCCAAGGCAGAACTCATCTTGAAGATTGTCTAGAGTGATTTTACCGCTGATG
AAATGAC
TGGACTCCCTCCTCCTGCTCTTATACGAAAAATTGCCTGACTCTGCAAAGGTTGTTTGTCTTGGAAGATGATGTGCCCC
CCCATCGC
TCTTATCTCATACCCCGCCATCTTTCTAGATTCTCATCTTCAACAAGAGGGGCAATCCATGATCTGCGATCCAGATGTG
CTTCTGGC
CTCATACTCTGCCTTCAGGTTGATGTTCACTTAATTGGTGACGAATTCAGCTGATTTGCTGCAGTATGCTTTGTGTTGG
TTCTTTCC
AGGCTTGTGC CAGCCATGAGCGCTTTGAGAGCATGTTGTCAC CTATAAACTC GAGTAAC
GGCCACATATTGTTCACTACTTGAATC
ACATACCTAATTTTGATAGAATTGACATGTTTAAAGAGCTGAGGTAGCTTTAATGCCTCTGAAGTATTGTGACACAGCT
TCTCACA
GAGTGAGAATGAAAAGTTGGACTCCCCCTAATGAAGTAAAAGTTTCGTCTCTGAACGGTGAAGAGCATAGATCCGGCAT
CAACTA
CCTGGCTAGACTACGACGTCAATTCTGC GGCCTTTTGAC
CTTTATATATGTCCATTAATGCAATAGATTCTTTTTTTTTTTTTTTTTT
TTTTTTTTTTTTTTTTTTTTTTTGCCCAATTTC GCAGATCAAAGTGGAC
GTTATAGCATCATAACTAAGCTCAGTTGCTGAGGGAAG
CCGTCTACTAC CTTAGC CCATC CAT CCAGCTC CATAC
CTTGATACTTTAGACGTGAAGCAATTCACACTGTAC GTCTCGCAGCTCTC
CTTCCCGCTCTTGCTTCC CCACTGGGGTCCATGGTGCGTGTATC GTCC CCTC
CTTAATTAAGGCCATTTAGGCCGTTGCTGGCGTTT
TT CCATAGGC TC CGCCCC
CCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGA
TAC CAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGAC CCTGC CGCTTACCGGATACCTGTC
CGCCTTTCTC CCT
TCGGGAAGCGT GGCGCTTTCT CATAGC TCACGC TGTAGGTAT CT CAGTTC GGTGTAGGTC GTTC
GCTCCAAGCTGGGCTGTGTGCA
CGAAC CCCCC GTTCAGCCC GACC GCTGCGC CTTATCCGGTAACTATCGTCTTGAGTCCAACCC
GGTAAGACACGACTTATCGC CAC
TGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAA
CTACGG
CTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGA
TCCGGCA
AACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGA
TCCTTT
GATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGCCTGCAGGGCCGATTTTGGTCATGAGAT
TATCAA
AAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTC
TGACAGT
TAC CAATGC TTAAT CAGTGAGGCACC TATC TCAGCGAT CT GTCTATTTCGTTCAT CCATAGTTGCC
TGAC TCCC CGTCGTGTAGATA
ACTACGATACGGGAGGGCTTACCATCTGGC CCCAGTGCTGCAATGATACC GCGAGAC CCACGCTCACC GGCTCC
AGATTTATC AG
CAATAAACCAGC CAGCCGGAAGGGC CGAGCGCAGAAGTGGTCCTGCAACTTTATCC
GCCTCCATCCAGTCTATTAATTGTTGC CG
GGAAGCTAGAGTAAGTAGTTC GCCAGTTAATAGTTTGCGCAAC
GTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGT
TT GGTAT GGCTT CATT CAGCTC CGGTTCCCAAC GATCAAGGCGAGTTACATGATCC CCCAT
GTTGTGCAAAAAA GCGGTTAGCT CC
TT CGGTC CTCC
GATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTC
ATGCCATCC GTAAGATGCTTTTCTGTGACTGGTGAGTACTCAAC CAAGTCATTCTGAGAATAGTGTATGC
GGCGACC GAGTTGCTC
TT GCCCGGCGTCAATACGGGATAATACC
GCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGC GA
AAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTT
TTACTTTC
ACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAA
TACTC
ATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGGCCATTTAGGCCT (Seq ID no 10)
[00294] SEQ ID NO: 11 = Nucleotide sequence of the pTrex8gM TR1033 expression construct.
CTAGAGTTGTGAAGTC GGTAATC CCGCTGTATAGTAATAC GAGTCGCATCTAAATACTCC
GAAGCTGCTGCGAACCCGGAGAATC
GAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAA
TCATGG
CGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTC CCGAAAAAACTC GGAGATTC
CTAAGTAGCGAT
GGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAAC
TGTTCC
GTAC CCCACCTCTTCTCAACCTTTGGC GTTT CC CTGATTCAGC GTACC
CGTACAAGTCGTAATCACTATTAACCCAGACTGACC GG
ACGTGTTTTGC CCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGAC
CGACTGGGGCTGTTC GAAGCCCGA
ATGTAGGATTGTTATC CGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGC
CTCGAAGGTTCACGGCAA
GGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCC GCAATGCAGCATCACTGGAAAATACAAAC
CAATGGCTAAAA
GTACATAAGTTAATGC CTAAAGAAGTCATATAC
CAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAA
CGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGG
TGATCCC
CCAATTGGGTCGCTTGTTTGTTCC GGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTC
GGAGCGTTTTGCATACAACCAA
GGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAG
GTTTGT
CTGCCGATACGACGAATACTGTATAGTCACTTCTGATGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCA
AAAGAT
TGAGTTGAAAC TGCC TAAGAT CT CGGGCCCT CGGGCC TTC GGCCTTTGGGTGTACATGTTTGTGCTCC
GGGCAAATGCAAAGTGTG
GTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGT
TTCGAA
TAGAAAGAGAAGCTTAGCCAAGAACAATAGCCGATAAAGATAGCCTCATTAAACGGAATGAGCTAGTAGGCAAAGTCAG
CGAAT
GTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGAC
TTGTACA
CCATCTTTTGAGGCACAGAAACCCAATAGTCAACCATCACAAGTTTGTACAAAAAAGCAGGCTTCACCATGCAGACCTT
CGGTGC
TTTTCTCGTTTCCTTCCTCGCCGCCAGGTAAGTTGGCCTTGATGAACCATATCATATATCGCCGAGAAGTGGACCGCGT
GCTGAGA
CTGAGACAGCGGCCTGGCCGCGGCCACCAAGAAGCCCCTCGTCAACGAGCTGAAGCTCCAGAAGGACATCAACATCAAG
GACCT
CATGGCTGGCGCCCAGAAGCTCCAGGACATTGCCGAGGCCAACGGCAACACCCGCGTCTTTGGCGGCGCTGGCCACAAC
GCCACC
GTCGACTACCTCTACAAGACCCTCAAGGCCACCGGCTACTACAACGTCAAGAAGCAGCCCTTCACCGAGCTGTACAGCG
CCGGCA
CCGCCAGCCTCAAGGTCGACGGCGACGACATCACCGCCGCCATCATGACCTACACCCCTGCCGGCGAGGCCACCGGCCC
TCTTGT
CGTCGCTGAGAACCTTGGCTGCGAGGCCAGCGACTTCCCCGCTGAGTCTGAGGGCAAGGTCGTCCTCGTCCTCCGCGGC
GAGTGC
CCCTTCAGCCAGAAGTCCACCAACGGCAAGACTGCCGGCGCTGCCGCCGTCATCGTCTACAACAACGTCCCCGGCGAGC
TGGCCG
GCACTCTCGGCGAACCCTTTGGCGAGTTCGCCCCCATCGTCGGCATCAGCCAAGAGGACGGCCAGGCCATCCTCGCCAA
GACCAA
GGCCGGCGAGGTCACGGTCGACCTGAAGGTCGACGCCACGGTCGAGAACCGCGTCACCTTCAACGTCATTGCCGAGACT
AAGGA
AGGCGACCACGACAACGTCCTCGTCGTCGGCGGCCACTCTGATAGCGTCGCTGCCGGCCCTGGCATCAACGACGACGGC
AGCGGC
ATCATCGGCATCCTCAAGGTCGCCCAGGCCCTCACCAAGTACCGCGTCAAGAACGCCGTCCGCTTCGGCTTCTGGTCCG
CCGAAG
AGTTCGGCCTCCTCGGCAGCTACGCCTACATGAAGTCGATCAACGGCTCCGACGCCGAGGTCGCCAAGATCCGCGCCTA
CCTCAA
CTTCGACATGATCGCCAGCCCCAACTACGTCTACGGCATCTACGACGGCGACGGCAGCGCCTTCAACCTCACTGGCCCT
GCCGGC
TCGGACGCCATCGAGAAGGACTTCGAGCGCTTCTTCAAGACCAAGCGCCTCGGCTACGTCCCCAGCGAGTTTAGCGGCC
GCTCTG
ACTACGCCGCCTTCATCGAGAACGGCATCCCCAGCGGCGGACTCTTCACTGGCGCCGAGCAGCTCAAGACCGAGGAAGA
GGCCA
AGAAGTTCGGCGGCGAGGCCGGCGTCGCCTACGACATCAACTACCACAAGATCGGCGACGATATCAACAACCTCAACAA
GGAAG
CCTTCCTCGTCAACACCCAGGCCATTGCCAACAGCGTCGCCCGCTACGCCAAGACCTGGAAGTCCCTGCCCAAGGTCAC
CCACAA
CACCCGCCGATGGGACGCCGAGGTTGCCTCCGTCCTCAAGCGAAGCAGCGGCCACTCTCACGCTGGCGGCCCTTGTGGC
TCTGTC
AGCGTCTAAGACCCAGCTTTCTTGTACAAAGTGGTGATCGCGCCAGCTCCGTGCGAAAGCCTGACGCACCGGTAGATTC
TTGGTG
AGCCCGTATCATGACGGCGGCGGGAGCTACATGGCCCCGGGTGATTTATTTTTTTTGTATCTACTTCTGACCCTTTTCA
AATATACG
GTCAACTCATCTTTCACTGGAGATGCGGCCTGCTTGGTATTGCGATGTTGTCAGCTTGGCAAATTGTGGCTTTCGAAAA
CACAAAA
CGATTCCTTAGTAGCCATGCATTTTAAGATAACGGAATAGAAGAAAGAGGAAATTAAAAAAAAAAAAAAAACAAACATC
CCGTT
CATAACCCGTAGAATCGCCGCTCTTCGGCTAGCTAGTTACGCTTGTTTATTTACGACAAGATCTAGAAGATTCGAGATA
GAATAAT
AATAATAACAACAATTTGCCTCTTCTTTCCACCTTTTCAGTCTTACTCTCCCTTCTGACATTGAACGCCTCAATCAGTC
AGTCGCCT
TGTACTTGGCACGGTAATCCTCCGTGTTCTTGATATCCTCAGGGGTAGCAAAGCCCTTCATGCCATCGATAATGTCATC
CAGAGTG
AGGATGGCAAAGATGGGGATGCCGTACTCCTTCCTCAGCTCGCCAATGGCACTCGGTCCAGGCTTGGAGTCGTCGCCAT
CCGCAG
CGGGGAGCTTCTCCATGCGGTCCAGGGCCACGACGATGCCGGCGACGATGCCGCCCTCCTTGGTGATCTTCTCAATGGC
GTCCCTC
TTGGCGGTGCCGGCGGTGATGACGTCGTCGACAATCAGGACCCTCTTGCCCTTGAGCGAAGCGCCGACGATGTTGCCGC
CCTCGC
CGTGGTCCTTGGCCTCCTTGCGGTCAAACGAGTAGGAGACGCGGTCCAGGTTCTGGGGCGCCAGCTCGCCGAGCTTGAT
GGTGAT
GGCGGAGCACAGCGGGATGCCCTTGTAGGCCGGGCCGAAGACGATGTCGAACTCTAGGCCGGCCTTCTCCTGGGCCTCG
ATGATG
GTCTTTGCAAAGGCGGAGGCGATGGCGCCGGCGAGGCGCGCCGTGTGGAATTCGCCCGCGTTGAAGAAGTAGGGGGATA
TCCGC
TTGGACTTGAGCTCGAAGCTGCCAAACTTGAGGACGCCGCCGTCGATGGCGGATTTGAGGAAGTCCTGCTTGTAGGCAG
GCAGCT
GGGAGGTGGTAGCCATTCTGTTGGATTTGGATAGTGTCCTTATTCTCTGATTTGAACAGTAGATCAGGACGAGTGAGAG
GGATGC
AGAGGTTGGATTGGAGTGGTTGAGCTATAAAATTTAGAGGCGCGCCGTATCGAGTTTTCACATGGAAGTCAAAGCGTAC
AGTGCG
AGCTTGTACGTTGGTCTTAGTATCCCACAAGCTTCTGTCTAGGTATGATGATGGCTATAAGTCACCCAAGGCAGAACTC
ATCTTGA
AGATTGTCTAGAGTGATTTTACCGCTGATGAAATGACTGGACTCCCTCCTCCTGCTCTTATACGAAAAATTGCCTGACT
CTGCAAA
GGTTGTTTGTCTTGGAAGATGATGTGCCCCCCCATCGCTCTTATCTCATACCCCGCCATCTTTCTAGATTCTCATCTTC
AACAAGAG
GGGCAATCCATGATCTGCGATCCAGATGTGCTTCTGGCCTCATACTCTGCCTTCAGGTTGATGTTCACTTAATTGGTGA
CGAATTC
AGCTGATTTGCTGCAGTATGCTTTGTGTTGGTTCTTTCCAGGCTTGTGCCAGCCATGAGCGCTTTGAGAGCATGTTGTC
ACCTATAA
ACTCGAGTAACGGCCACATATTGTTCACTACTTGAATCACATACCTAATTTTGATAGAATTGACATGTTTAAAGAGCTG
AGGTAGC
TTTAATGCCTCTGAAGTATTGTGACACAGCTTCTCACAGAGTGAGAATGAAAAGTTGGACTCCCCCTAATGAAGTAAAA
GTTTCGT
CTCTGAACGGTGAAGAGCATAGATCCGGCATCAACTACCTGGCTAGACTACGACGTCAATTCTGCGGCCTTTTGACCTT
TATATAT
GTCCATTAATGCAATAGATTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCCCAATTTCGCAGATC
AAAGTGGACGT
TATAGCATCATAACTAAGCTCAGTTGCTGAGGGAAGCCGTCTACTACCTTAGCC CATC CATCCA GCT CCATACC
TT GATACTTTAG
ACGTGAAGCAATTCACACTGTACGTCTCGCAGCTCTC CTTC CC GCTCTTGCTTCCC CACTGGGGTCCATGGTGC
GTGTATC GTCCCC
TCCTTAATTAAGGCCATTTAGGCCGTTGCTGGC GTTTTTCCATAGGCTCC GCCCC CCTGAC
GAGCATCACAAAAATC GACGCT CAA
GTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGC GTTTCCC CCTGGAAGCTCC CTC
GTGCGCTCTCCTGTTCC GAC
CCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTAT
CTCAGTTC
GGTGTAGGTC GTTCGCTCCAAGCTGGGCTGTGTGCAC GAACCCC CCGTTCAGC CCGACCGCTGCGC CTTATC
CGGTAACTATC GTC
TT GAGTCC AACCC GGTAAGACACGACTTATCGC CACTGGCAGCAGC
CACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCG
GTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAA
GCCAGTT
ACCTTCGGAAAAAGAGTT GGTA GC TCTT GATCC GGCAAACAAACCACC GCTGGTAGCGGT
GGTTTTTTTGTTT GCAAGCAGC AGA
TTACGCGCAGAAAAAAAGGATCTCAAGAAGATC
CTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCAC GTTA
AGGCCTGCAGGGCCGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTT
TTAAATC
AATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGT
CTATTTC
GTTCATC CATAGTTGCCTGACTCC CCGTC GTGTAGATAACTACGATACGGGAGGGCTTAC CATCTGGCCC
CAGTGCTGCAATGATA
CCGC GAGACCCACGCTCAC CGGCTCCAGATTTATCAGCAATAAACCAGC
CAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCA
ACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCA
ACGTTGT
TGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTC
CCAACGATCAAGGC GAGTTA
CAT GATCCC CCATGTTGTGCAAAAAAGC GGTTAGCTCCTTC GGTCCT CC
GATCGTTGTCAGAAGTAAGTTGGCC GCAGTGTTAT CA
CTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACT
CAACCAA
GTCATTCTGAGAATAGTGTATGC GGCGACCGAGTTGCTCTTGCCC
GGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACT
TTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTAC CGCTGTTGAGATCCAGTTC
GATGTAAC C
CACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAAT
GCCGCA
AAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGG
GTTATTG
TCTCATGGCCATTTAGGCCT (Seq ID no 11)
CTAGAGTTGTGAAGTC GGTAATC CCGCTGTATAGTAATAC GAGTCGCATCTAAATACTCC
GAAGCTGCTGCGAACCCGGAGAATC
GAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAA
TCATGG
CGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTC CCGAAAAAACTC GGAGATTC
CTAAGTAGCGAT
GGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAAC
TGTTCC
GTAC CCCACCTCTTCTCAACCTTTGGC GTTT CC CTGATTCAGC GTACC
CGTACAAGTCGTAATCACTATTAACCCAGACTGACC GG
ACGTGTTTTGC CCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGAC
CGACTGGGGCTGTTC GAAGCCCGA
ATGTAGGATTGTTATC CGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGC
CTCGAAGGTTCACGGCAA
GGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCC GCAATGCAGCATCACTGGAAAATACAAAC
CAATGGCTAAAA
GTACATAAGTTAATGC CTAAAGAAGTCATATAC
CAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAA
CGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGG
TGATCCC
CCAATTGGGTCGCTTGTTTGTTCC GGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTC
GGAGCGTTTTGCATACAACCAA
GGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAG
GTTTGT
CTGCCGATACGACGAATACTGTATAGTCACTTCTGATGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCA
AAAGAT
TGAGTTGAAAC TGCC TAAGAT CT CGGGCCCT CGGGCC TTC GGCCTTTGGGTGTACATGTTTGTGCTCC
GGGCAAATGCAAAGTGTG
GTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGT
TTCGAA
TAGAAAGAGAAGCTTAGCCAAGAACAATAGCCGATAAAGATAGCCTCATTAAACGGAATGAGCTAGTAGGCAAAGTCAG
CGAAT
GTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGAC
TTGTACA
CCATCTTTTGAGGCACAGAAACCCAATAGTCAACCATCACAAGTTTGTACAAAAAAGCAGGCTTCACCATGCAGACCTT
CGGTGC
TTTTCTCGTTTCCTTCCTCGCCGCCAGGTAAGTTGGCCTTGATGAACCATATCATATATCGCCGAGAAGTGGACCGCGT
GCTGAGA
CTGAGACAGCGGCCTGGCCGCGGCCACCAAGAAGCCCCTCGTCAACGAGCTGAAGCTCCAGAAGGACATCAACATCAAG
GACCT
CATGGCTGGCGCCCAGAAGCTCCAGGACATTGCCGAGGCCAACGGCAACACCCGCGTCTTTGGCGGCGCTGGCCACAAC
GCCACC
GTCGACTACCTCTACAAGACCCTCAAGGCCACCGGCTACTACAACGTCAAGAAGCAGCCCTTCACCGAGCTGTACAGCG
CCGGCA
CCGCCAGCCTCAAGGTCGACGGCGACGACATCACCGCCGCCATCATGACCTACACCCCTGCCGGCGAGGCCACCGGCCC
TCTTGT
CGTCGCTGAGAACCTTGGCTGCGAGGCCAGCGACTTCCCCGCTGAGTCTGAGGGCAAGGTCGTCCTCGTCCTCCGCGGC
GAGTGC
CCCTTCAGCCAGAAGTCCACCAACGGCAAGACTGCCGGCGCTGCCGCCGTCATCGTCTACAACAACGTCCCCGGCGAGC
TGGCCG
GCACTCTCGGCGAACCCTTTGGCGAGTTCGCCCCCATCGTCGGCATCAGCCAAGAGGACGGCCAGGCCATCCTCGCCAA
GACCAA
GGCCGGCGAGGTCACGGTCGACCTGAAGGTCGACGCCACGGTCGAGAACCGCGTCACCTTCAACGTCATTGCCGAGACT
AAGGA
AGGCGACCACGACAACGTCCTCGTCGTCGGCGGCCACTCTGATAGCGTCGCTGCCGGCCCTGGCATCAACGACGACGGC
AGCGGC
ATCATCGGCATCCTCAAGGTCGCCCAGGCCCTCACCAAGTACCGCGTCAAGAACGCCGTCCGCTTCGGCTTCTGGTCCG
CCGAAG
AGTTCGGCCTCCTCGGCAGCTACGCCTACATGAAGTCGATCAACGGCTCCGACGCCGAGGTCGCCAAGATCCGCGCCTA
CCTCAA
CTTCGACATGATCGCCAGCCCCAACTACGTCTACGGCATCTACGACGGCGACGGCAGCGCCTTCAACCTCACTGGCCCT
GCCGGC
TCGGACGCCATCGAGAAGGACTTCGAGCGCTTCTTCAAGACCAAGCGCCTCGGCTACGTCCCCAGCGAGTTTAGCGGCC
GCTCTG
ACTACGCCGCCTTCATCGAGAACGGCATCCCCAGCGGCGGACTCTTCACTGGCGCCGAGCAGCTCAAGACCGAGGAAGA
GGCCA
AGAAGTTCGGCGGCGAGGCCGGCGTCGCCTACGACATCAACTACCACAAGATCGGCGACGATATCAACAACCTCAACAA
GGAAG
CCTTCCTCGTCAACACCCAGGCCATTGCCAACAGCGTCGCCCGCTACGCCAAGACCTGGAAGTCCCTGCCCAAGGTCAC
CCACAA
CACCCGCCGATGGGACGCCGAGGTTGCCTCCGTCCTCAAGCGAAGCAGCGGCCACTCTCACGCTGGCGGCCCTTGTGGC
TCTGTC
AGCGTCTAAGACCCAGCTTTCTTGTACAAAGTGGTGATCGCGCCAGCTCCGTGCGAAAGCCTGACGCACCGGTAGATTC
TTGGTG
AGCCCGTATCATGACGGCGGCGGGAGCTACATGGCCCCGGGTGATTTATTTTTTTTGTATCTACTTCTGACCCTTTTCA
AATATACG
GTCAACTCATCTTTCACTGGAGATGCGGCCTGCTTGGTATTGCGATGTTGTCAGCTTGGCAAATTGTGGCTTTCGAAAA
CACAAAA
CGATTCCTTAGTAGCCATGCATTTTAAGATAACGGAATAGAAGAAAGAGGAAATTAAAAAAAAAAAAAAAACAAACATC
CCGTT
CATAACCCGTAGAATCGCCGCTCTTCGGCTAGCTAGTTACGCTTGTTTATTTACGACAAGATCTAGAAGATTCGAGATA
GAATAAT
AATAATAACAACAATTTGCCTCTTCTTTCCACCTTTTCAGTCTTACTCTCCCTTCTGACATTGAACGCCTCAATCAGTC
AGTCGCCT
TGTACTTGGCACGGTAATCCTCCGTGTTCTTGATATCCTCAGGGGTAGCAAAGCCCTTCATGCCATCGATAATGTCATC
CAGAGTG
AGGATGGCAAAGATGGGGATGCCGTACTCCTTCCTCAGCTCGCCAATGGCACTCGGTCCAGGCTTGGAGTCGTCGCCAT
CCGCAG
CGGGGAGCTTCTCCATGCGGTCCAGGGCCACGACGATGCCGGCGACGATGCCGCCCTCCTTGGTGATCTTCTCAATGGC
GTCCCTC
TTGGCGGTGCCGGCGGTGATGACGTCGTCGACAATCAGGACCCTCTTGCCCTTGAGCGAAGCGCCGACGATGTTGCCGC
CCTCGC
CGTGGTCCTTGGCCTCCTTGCGGTCAAACGAGTAGGAGACGCGGTCCAGGTTCTGGGGCGCCAGCTCGCCGAGCTTGAT
GGTGAT
GGCGGAGCACAGCGGGATGCCCTTGTAGGCCGGGCCGAAGACGATGTCGAACTCTAGGCCGGCCTTCTCCTGGGCCTCG
ATGATG
GTCTTTGCAAAGGCGGAGGCGATGGCGCCGGCGAGGCGCGCCGTGTGGAATTCGCCCGCGTTGAAGAAGTAGGGGGATA
TCCGC
TTGGACTTGAGCTCGAAGCTGCCAAACTTGAGGACGCCGCCGTCGATGGCGGATTTGAGGAAGTCCTGCTTGTAGGCAG
GCAGCT
GGGAGGTGGTAGCCATTCTGTTGGATTTGGATAGTGTCCTTATTCTCTGATTTGAACAGTAGATCAGGACGAGTGAGAG
GGATGC
AGAGGTTGGATTGGAGTGGTTGAGCTATAAAATTTAGAGGCGCGCCGTATCGAGTTTTCACATGGAAGTCAAAGCGTAC
AGTGCG
AGCTTGTACGTTGGTCTTAGTATCCCACAAGCTTCTGTCTAGGTATGATGATGGCTATAAGTCACCCAAGGCAGAACTC
ATCTTGA
AGATTGTCTAGAGTGATTTTACCGCTGATGAAATGACTGGACTCCCTCCTCCTGCTCTTATACGAAAAATTGCCTGACT
CTGCAAA
GGTTGTTTGTCTTGGAAGATGATGTGCCCCCCCATCGCTCTTATCTCATACCCCGCCATCTTTCTAGATTCTCATCTTC
AACAAGAG
GGGCAATCCATGATCTGCGATCCAGATGTGCTTCTGGCCTCATACTCTGCCTTCAGGTTGATGTTCACTTAATTGGTGA
CGAATTC
AGCTGATTTGCTGCAGTATGCTTTGTGTTGGTTCTTTCCAGGCTTGTGCCAGCCATGAGCGCTTTGAGAGCATGTTGTC
ACCTATAA
ACTCGAGTAACGGCCACATATTGTTCACTACTTGAATCACATACCTAATTTTGATAGAATTGACATGTTTAAAGAGCTG
AGGTAGC
TTTAATGCCTCTGAAGTATTGTGACACAGCTTCTCACAGAGTGAGAATGAAAAGTTGGACTCCCCCTAATGAAGTAAAA
GTTTCGT
CTCTGAACGGTGAAGAGCATAGATCCGGCATCAACTACCTGGCTAGACTACGACGTCAATTCTGCGGCCTTTTGACCTT
TATATAT
GTCCATTAATGCAATAGATTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCCCAATTTCGCAGATC
AAAGTGGACGT
TATAGCATCATAACTAAGCTCAGTTGCTGAGGGAAGCCGTCTACTACCTTAGCC CATC CATCCA GCT CCATACC
TT GATACTTTAG
ACGTGAAGCAATTCACACTGTACGTCTCGCAGCTCTC CTTC CC GCTCTTGCTTCCC CACTGGGGTCCATGGTGC
GTGTATC GTCCCC
TCCTTAATTAAGGCCATTTAGGCCGTTGCTGGC GTTTTTCCATAGGCTCC GCCCC CCTGAC
GAGCATCACAAAAATC GACGCT CAA
GTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGC GTTTCCC CCTGGAAGCTCC CTC
GTGCGCTCTCCTGTTCC GAC
CCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTAT
CTCAGTTC
GGTGTAGGTC GTTCGCTCCAAGCTGGGCTGTGTGCAC GAACCCC CCGTTCAGC CCGACCGCTGCGC CTTATC
CGGTAACTATC GTC
TT GAGTCC AACCC GGTAAGACACGACTTATCGC CACTGGCAGCAGC
CACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCG
GTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAA
GCCAGTT
ACCTTCGGAAAAAGAGTT GGTA GC TCTT GATCC GGCAAACAAACCACC GCTGGTAGCGGT
GGTTTTTTTGTTT GCAAGCAGC AGA
TTACGCGCAGAAAAAAAGGATCTCAAGAAGATC
CTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCAC GTTA
AGGCCTGCAGGGCCGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTT
TTAAATC
AATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGT
CTATTTC
GTTCATC CATAGTTGCCTGACTCC CCGTC GTGTAGATAACTACGATACGGGAGGGCTTAC CATCTGGCCC
CAGTGCTGCAATGATA
CCGC GAGACCCACGCTCAC CGGCTCCAGATTTATCAGCAATAAACCAGC
CAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCA
ACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCA
ACGTTGT
TGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTC
CCAACGATCAAGGC GAGTTA
CAT GATCCC CCATGTTGTGCAAAAAAGC GGTTAGCTCCTTC GGTCCT CC
GATCGTTGTCAGAAGTAAGTTGGCC GCAGTGTTAT CA
CTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACT
CAACCAA
GTCATTCTGAGAATAGTGTATGC GGCGACCGAGTTGCTCTTGCCC
GGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACT
TTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTAC CGCTGTTGAGATCCAGTTC
GATGTAAC C
CACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAAT
GCCGCA
AAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGG
GTTATTG
TCTCATGGCCATTTAGGCCT (Seq ID no 11)
[00295] SEQ ID NO: 12 = Nucleotide sequence of the pTrex8gM TR1034 expression construct.
CTAGAGTTGTGAAGTC GGTAATC CCGCTGTATAGTAATAC GAGTCGCATCTAAATACTCC
GAAGCTGCTGCGAACCCGGAGAATC
GAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAA
TCATGG
CGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTC CCGAAAAAACTC GGAGATTC
CTAAGTAGCGAT
GGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAAC
TGTTCC
GTAC CCCACCTCTTCTCAACCTTTGGC GTTT CC CTGATTCAGC GTACC
CGTACAAGTCGTAATCACTATTAACCCAGACTGACC GG
ACGTGTTTTGC CCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGAC
CGACTGGGGCTGTTC GAAGCCCGA
ATGTAGGATTGTTATC CGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGC
CTCGAAGGTTCACGGCAA
GGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCC GCAATGCAGCATCACTGGAAAATACAAAC
CAATGGCTAAAA
GTACATAAGTTAATGC CTAAAGAAGTCATATAC
CAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAA
CGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGG
TGATCCC
CCAATTGGGTCGCTTGTTTGTTCC GGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTC
GGAGCGTTTTGCATACAACCAA
GGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAG
GTTTGT
CTGCCGATACGACGAATACTGTATAGTCACTTCTGATGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCA
AAAGAT
TGAGTTGAAAC TGCC TAAGAT CT CGGGCCCT CGGGCC TTC GGCCTTTGGGTGTACATGTTTGTGCTCC
GGGCAAATGCAAAGTGTG
GTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGT
TTCGAA
TAGAAAGAGAAGCTTAGCCAAGAACAATAGC CGATAAAGATAGC CTCATTAAAC
GGAATGAGCTAGTAGGCAAAGTCAGCGAAT
GTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGAC
TTGTACA
CCATCTTTTGAGGCACAGAAACCCAATAGTCAAC CATCACAAGTTTGTACAAAAAAGCAGGCTTCACCATGCAGAC
CTTCGGTGC
TTTTCTCGTTTCCTTCCTCGCCGCCAGGTAAGTTGGCCTTGATGAACCATATCATATATCGCCGAGAAGTGGACCGCGT
GCTGAGA
CTGAGACAGCGGCCTGGCCGCGGCCCTCCAGATTCCTCTCAACCTCCAGGTCCCCAAGCTCAGCTGGAACCTCTTCGGC
GACGAC
CTCCCCCTGGTCGACACCAAGGAACTCCAGAAGTCCATCAAGCCCGAGAACCTTGAGGCCCGAGCCAAGGACCTCTACG
AGATCG
CCAAGAACGGCGAGGAAGAGTACGGCCACCCCACCCGCGTCATTGGCTCTGAGGGCCACCTCGGCACCCTCAGCTACAT
CCACGC
CGAGCTGGCTAAGCTCGGCGGCTACTACAGCGTCAGCAACCAGCAGTTCCCCGCCGTCAGCGGCAACGTCTTTGAGAGC
CGCCTC
GTCATCGGCGACAGCGTCCCTAAGCAGGCCAGCCCTATGGGCCTCACCCCCCCCACCAAGAACAAGGAACCCGTCCACG
GCACCC
TCGTCCTCGTCGACAACGAGGGCTGCGACGCCAGCGACTACCCCGAGGCTGTCAAGGGCAACATTGCCCTCATCCTCCG
CGGCAC
GTGCCCCTTCGGCACCAAGTCTGGCAACGCCGGCAAGGCTGGCGCCGTCGCTGCTGTCGTCTACAACTACGAGAAGGAC
GAGGTC
CACGGCACGCTGGGCACCCCTAGCCCTGATCACGTCGCCACCTTTGGCCTCGGCGGCGAAGAGGGCAAGGCCGTCGCCA
AGAAG
CTCAAGGACGGCGAGAAGGTCGACGCCATTGCCTACATTGACGCCGAGGTCAAGACCATCAGCACCACCAACATCATTG
CCCAG
ACCCGAGGCGGCGACCCCGACAACTGCGTTATGCTTGGCGGCCACAGCGACAGCGTCGCTGAGGGCCCTGGCATCAACG
ACGAT
GGCAGCGGCAGCATCAGCGTCCTTGAGGTCGCCGTCCAGCTCACCAAGTACCGCGTCAACAACTGCGTCCGCTTCGCCT
GGTGGG
CCGCTGAGGAAGAGGGCCTCCTTGGCAGCGACCACTACGTCAGCGTCCTCCCCGAGGACGAGAACCGCAAGATCCGCCT
CTTCAT
GGACTACGACATGATGGCCAGCCCCAACTTCGCCTACCAGATCTACAACGCCACCAACGCCGAGAACCCCAAGGGCAGC
GAGGA
ACTCCGCGACCTCTACGTCAACTGGTACGAGGAACAGGGCCTCAACTACACCTTCATTCCCTTCGACGGCCGCAGCGAC
TACGAC
GGCTTTATCCGAGGCGGCATCCCCGCTGGCGGCATTGCTACTGGCGCTGAGGGCGTCAAGACCGAGGACGAGGTCGAGA
TGTTCG
GCGGCGAGGCCGGCGTCTGGTACGACAAGAACTACCACCAGATTGGCGACGACCTGACCAACGTCAACTACACCGCCTG
GGAGG
TCAACACCAAGCTGATCGCCCACAGCGTCGCCACCTACGCCAAGAGCTTCAAGGGCTTCCCCGAGCGCGAGATCGAGAC
TAGCGT
CCAGACCTACAGCGACAAGACCAAGTACCACGGCAGCAAGCTGTTCATCTAAGACCCAGCTTTCTTGTACAAAGTGGTG
ATCGCG
CCAGCTCCGTGCGAAAGCCTGACGCACCGGTAGATTCTTGGTGAGCCCGTATCATGACGGCGGCGGGAGCTACATGGCC
CCGGGT
GATTTATTTTTTTTGTATCTACTTCTGACCCTTTTCAAATATACGGTCAACTCATCTTTCACTGGAGATGCGGCCTGCT
TGGTATTGC
GATGTTGTCAGCTTGGCAAATTGTGGCTTTCGAAAACACAAAACGATTCCTTAGTAGCCATGCATTTTAAGATAACGGA
ATAGAA
GAAAGAGGAAATTAAAAAAAAAAAAAAAACAAACATCCCGTTCATAACCCGTAGAATCGCCGCTCTTCGGCTAGCTAGT
TACGC
TTGTTTATTTACGACAAGATCTAGAAGATTCGAGATAGAATAATAATAATAACAACAATTTGCCTCTTCTTTCCACCTT
TTCAGTCT
TACTCTCCCTTCTGACATTGAACGCCTCAATCAGTCAGTCGCCTTGTACTTGGCACGGTAATCCTCCGTGTTCTTGATA
TCCTCAGG
GGTAGCAAAGCCCTTCATGCCATCGATAATGTCATCCAGAGTGAGGATGGCAAAGATGGGGATGCCGTACTCCTTCCTC
AGCTCG
CCAATGGCACTCGGTCCAGGCTTGGAGTCGTCGCCATCCGCAGCGGGGAGCTTCTCCATGCGGTCCAGGGCCACGACGA
TGCCGG
CGACGATGCCGCCCTCCTTGGTGATCTTCTCAATGGCGTCCCTCTTGGCGGTGCCGGCGGTGATGACGTCGTCGACAAT
CAGGACC
CTCTTGCCCTTGAGCGAAGCGCCGACGATGTTGCCGCCCTCGCCGTGGTCCTTGGCCTCCTTGCGGTCAAACGAGTAGG
AGACGC
GGTCCAGGTTCTGGGGCGCCAGCTCGCCGAGCTTGATGGTGATGGCGGAGCACAGCGGGATGCCCTTGTAGGCCGGGCC
GAAGA
CGATGTCGAACTCTAGGCCGGCCTTCTCCTGGGCCTCGATGATGGTCTTTGCAAAGGCGGAGGCGATGGCGCCGGCGAG
GCGCGC
CGTGTGGAATTCGCCCGCGTTGAAGAAGTAGGGGGATATCCGCTTGGACTTGAGCTCGAAGCTGCCAAACTTGAGGACG
CCGCCG
TCGATGGCGGATTTGAGGAAGTCCTGCTTGTAGGCAGGCAGCTGGGAGGTGGTAGCCATTCTGTTGGATTTGGATAGTG
TCCTTAT
TCTCTGATTTGAACAGTAGATCAGGACGAGTGAGAGGGATGCAGAGGTTGGATTGGAGTGGTTGAGCTATAAAATTTAG
AGGCGC
GCCGTATCGAGTTTTCACATGGAAGTCAAAGCGTACAGTGCGAGCTTGTACGTTGGTCTTAGTATCCCACAAGCTTCTG
TCTAGGT
ATGATGATGGCTATAAGTCACCCAAGGCAGAACTCATCTTGAAGATTGTCTAGAGTGATTTTACCGCTGATGAAATGAC
TGGACT
CCCTCCTCCTGCTCTTATACGAAAAATTGCCTGACTCTGCAAAGGTTGTTTGTCTTGGAAGATGATGTGCCCCCCCATC
GCTCTTAT
CTCATACCCCGCCATCTTTCTAGATTCTCATCTTCAACAAGAGGGGCAATCCATGATCTGCGATCCAGATGTGCTTCTG
GCCTCAT
ACTCTGCCTTCAGGTTGATGTTCACTTAATTGGTGACGAATTCAGCTGATTTGCTGCAGTATGCTTTGTGTTGGTTCTT
TCCAGGCT
TGTGCCAGCCATGAGCGCTTTGAGAGCATGTTGTCACCTATAAACTCGAGTAACGGCCACATATTGTTCACTACTTGAA
TCACATA
CCTAATTTTGATAGAATTGACATGTTTAAAGAGCTGAGGTAGCTTTAATGCCTCTGAAGTATTGTGACACAGCTTCTCA
CAGAGTG
AGAATGAAAAGTTGGACTCCCCCTAATGAAGTAAAAGTTTCGTCTCTGAACGGTGAAGAGCATAGATCCGGCATCAACT
ACCTGG
CTAGACTACGACGTCAATTCTGCGGCCTTTTGACCTTTATATATGTCCATTAATGCAATAGATTCTTTTTTTTTTTTTT
TTTTTTTTTT
TTTTTTTTTTTTTTTTTGCCCAATTTCGCAGATCAAAGTGGACGTTATAGCATCATAACTAAGCTCAGTTGCTGAGGGA
AGCCGTCT
ACTACCTTAGCCCATCCATCCAGCTCCATACCTTGATACTTTAGACGTGAAGCAATTCACACTGTACGTCTCGCAGCTC
TCCTTCCC
GCTCTTGCTTCCCCACTGGGGTCCATGGTGCGTGTATCGTCCCCTCCTTAATTAAGGCCATTTAGGCCGTTGCTGGCGT
TTTTCCAT
AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGAT
ACCAG
GCGTTTCC CCCTGGAAGCTC CCTCGTGC GCTCTCCTGTTCC GAC CCTGC CGCTTACCGGATACCTGTCC
GCCTTTCTCCCTTCGGGA
AGCGTGGCGCTTTCTCATAGCTCAC GCTGTAGGTATCTCAGTTC GGTGTAGGTC GTTC
GCTCCAAGCTGGGCTGTGTGCACGAACC
CCC CGTTCAGCCC GACCGCT GCGCC TTAT CCGGTAACTAT CGTC TT GAGTCC AACC
CGGTAAGACACGACTTATC GCCACTGGCAG
CAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGG
CTACAC
TAGAAGGACAGTATTTGGTATCTGC GCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATC
CGGCAAAC AA
ACCACC GCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATC
CTTT GATC TT
TTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGCCTGCAGGGCCGATTTTGGTCATGAGATTATCAA
AAAGGA
TCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAG
TTACCAA
TGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGA
TAACTACG
ATAC GGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATAC CGCGAGAC CCACGCTCACC
GGCTCCAGATTTATCAGCAATAA
ACCAGC CAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGC CTC
CATCCAGTCTATTAATTGTTGC CGGGAAGC
TAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCG
TTTGGTA
TGGCTTCATTCAGCTCCGGTTC CCAACGATCAAGGC GAGTTACATGATCCC
CCATGTTGTGCAAAAAAGCGGTTAGCTC CTTCGGT
CCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTG
TCATGCC
ATCC GTAAGATGCTTTTCTGTGACTGGTGAGTACTCAAC
CAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCC
CGGCGTCAATACGGGATAATACC GCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAAC
GTTCTTCGGGGCGAAAACT
CTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACT
TTCACCAG
CGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCC GCAAAAAAGGGAATAAGGGC
GACACGGAAATGTTGAATACTCATACT
CTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGGCCATTTAGGCCT (Seq ID no 12)
CTAGAGTTGTGAAGTC GGTAATC CCGCTGTATAGTAATAC GAGTCGCATCTAAATACTCC
GAAGCTGCTGCGAACCCGGAGAATC
GAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAA
TCATGG
CGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTC CCGAAAAAACTC GGAGATTC
CTAAGTAGCGAT
GGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAAC
TGTTCC
GTAC CCCACCTCTTCTCAACCTTTGGC GTTT CC CTGATTCAGC GTACC
CGTACAAGTCGTAATCACTATTAACCCAGACTGACC GG
ACGTGTTTTGC CCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGAC
CGACTGGGGCTGTTC GAAGCCCGA
ATGTAGGATTGTTATC CGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGC
CTCGAAGGTTCACGGCAA
GGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCC GCAATGCAGCATCACTGGAAAATACAAAC
CAATGGCTAAAA
GTACATAAGTTAATGC CTAAAGAAGTCATATAC
CAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAA
CGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGG
TGATCCC
CCAATTGGGTCGCTTGTTTGTTCC GGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTC
GGAGCGTTTTGCATACAACCAA
GGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAG
GTTTGT
CTGCCGATACGACGAATACTGTATAGTCACTTCTGATGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCA
AAAGAT
TGAGTTGAAAC TGCC TAAGAT CT CGGGCCCT CGGGCC TTC GGCCTTTGGGTGTACATGTTTGTGCTCC
GGGCAAATGCAAAGTGTG
GTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGT
TTCGAA
TAGAAAGAGAAGCTTAGCCAAGAACAATAGC CGATAAAGATAGC CTCATTAAAC
GGAATGAGCTAGTAGGCAAAGTCAGCGAAT
GTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGAC
TTGTACA
CCATCTTTTGAGGCACAGAAACCCAATAGTCAAC CATCACAAGTTTGTACAAAAAAGCAGGCTTCACCATGCAGAC
CTTCGGTGC
TTTTCTCGTTTCCTTCCTCGCCGCCAGGTAAGTTGGCCTTGATGAACCATATCATATATCGCCGAGAAGTGGACCGCGT
GCTGAGA
CTGAGACAGCGGCCTGGCCGCGGCCCTCCAGATTCCTCTCAACCTCCAGGTCCCCAAGCTCAGCTGGAACCTCTTCGGC
GACGAC
CTCCCCCTGGTCGACACCAAGGAACTCCAGAAGTCCATCAAGCCCGAGAACCTTGAGGCCCGAGCCAAGGACCTCTACG
AGATCG
CCAAGAACGGCGAGGAAGAGTACGGCCACCCCACCCGCGTCATTGGCTCTGAGGGCCACCTCGGCACCCTCAGCTACAT
CCACGC
CGAGCTGGCTAAGCTCGGCGGCTACTACAGCGTCAGCAACCAGCAGTTCCCCGCCGTCAGCGGCAACGTCTTTGAGAGC
CGCCTC
GTCATCGGCGACAGCGTCCCTAAGCAGGCCAGCCCTATGGGCCTCACCCCCCCCACCAAGAACAAGGAACCCGTCCACG
GCACCC
TCGTCCTCGTCGACAACGAGGGCTGCGACGCCAGCGACTACCCCGAGGCTGTCAAGGGCAACATTGCCCTCATCCTCCG
CGGCAC
GTGCCCCTTCGGCACCAAGTCTGGCAACGCCGGCAAGGCTGGCGCCGTCGCTGCTGTCGTCTACAACTACGAGAAGGAC
GAGGTC
CACGGCACGCTGGGCACCCCTAGCCCTGATCACGTCGCCACCTTTGGCCTCGGCGGCGAAGAGGGCAAGGCCGTCGCCA
AGAAG
CTCAAGGACGGCGAGAAGGTCGACGCCATTGCCTACATTGACGCCGAGGTCAAGACCATCAGCACCACCAACATCATTG
CCCAG
ACCCGAGGCGGCGACCCCGACAACTGCGTTATGCTTGGCGGCCACAGCGACAGCGTCGCTGAGGGCCCTGGCATCAACG
ACGAT
GGCAGCGGCAGCATCAGCGTCCTTGAGGTCGCCGTCCAGCTCACCAAGTACCGCGTCAACAACTGCGTCCGCTTCGCCT
GGTGGG
CCGCTGAGGAAGAGGGCCTCCTTGGCAGCGACCACTACGTCAGCGTCCTCCCCGAGGACGAGAACCGCAAGATCCGCCT
CTTCAT
GGACTACGACATGATGGCCAGCCCCAACTTCGCCTACCAGATCTACAACGCCACCAACGCCGAGAACCCCAAGGGCAGC
GAGGA
ACTCCGCGACCTCTACGTCAACTGGTACGAGGAACAGGGCCTCAACTACACCTTCATTCCCTTCGACGGCCGCAGCGAC
TACGAC
GGCTTTATCCGAGGCGGCATCCCCGCTGGCGGCATTGCTACTGGCGCTGAGGGCGTCAAGACCGAGGACGAGGTCGAGA
TGTTCG
GCGGCGAGGCCGGCGTCTGGTACGACAAGAACTACCACCAGATTGGCGACGACCTGACCAACGTCAACTACACCGCCTG
GGAGG
TCAACACCAAGCTGATCGCCCACAGCGTCGCCACCTACGCCAAGAGCTTCAAGGGCTTCCCCGAGCGCGAGATCGAGAC
TAGCGT
CCAGACCTACAGCGACAAGACCAAGTACCACGGCAGCAAGCTGTTCATCTAAGACCCAGCTTTCTTGTACAAAGTGGTG
ATCGCG
CCAGCTCCGTGCGAAAGCCTGACGCACCGGTAGATTCTTGGTGAGCCCGTATCATGACGGCGGCGGGAGCTACATGGCC
CCGGGT
GATTTATTTTTTTTGTATCTACTTCTGACCCTTTTCAAATATACGGTCAACTCATCTTTCACTGGAGATGCGGCCTGCT
TGGTATTGC
GATGTTGTCAGCTTGGCAAATTGTGGCTTTCGAAAACACAAAACGATTCCTTAGTAGCCATGCATTTTAAGATAACGGA
ATAGAA
GAAAGAGGAAATTAAAAAAAAAAAAAAAACAAACATCCCGTTCATAACCCGTAGAATCGCCGCTCTTCGGCTAGCTAGT
TACGC
TTGTTTATTTACGACAAGATCTAGAAGATTCGAGATAGAATAATAATAATAACAACAATTTGCCTCTTCTTTCCACCTT
TTCAGTCT
TACTCTCCCTTCTGACATTGAACGCCTCAATCAGTCAGTCGCCTTGTACTTGGCACGGTAATCCTCCGTGTTCTTGATA
TCCTCAGG
GGTAGCAAAGCCCTTCATGCCATCGATAATGTCATCCAGAGTGAGGATGGCAAAGATGGGGATGCCGTACTCCTTCCTC
AGCTCG
CCAATGGCACTCGGTCCAGGCTTGGAGTCGTCGCCATCCGCAGCGGGGAGCTTCTCCATGCGGTCCAGGGCCACGACGA
TGCCGG
CGACGATGCCGCCCTCCTTGGTGATCTTCTCAATGGCGTCCCTCTTGGCGGTGCCGGCGGTGATGACGTCGTCGACAAT
CAGGACC
CTCTTGCCCTTGAGCGAAGCGCCGACGATGTTGCCGCCCTCGCCGTGGTCCTTGGCCTCCTTGCGGTCAAACGAGTAGG
AGACGC
GGTCCAGGTTCTGGGGCGCCAGCTCGCCGAGCTTGATGGTGATGGCGGAGCACAGCGGGATGCCCTTGTAGGCCGGGCC
GAAGA
CGATGTCGAACTCTAGGCCGGCCTTCTCCTGGGCCTCGATGATGGTCTTTGCAAAGGCGGAGGCGATGGCGCCGGCGAG
GCGCGC
CGTGTGGAATTCGCCCGCGTTGAAGAAGTAGGGGGATATCCGCTTGGACTTGAGCTCGAAGCTGCCAAACTTGAGGACG
CCGCCG
TCGATGGCGGATTTGAGGAAGTCCTGCTTGTAGGCAGGCAGCTGGGAGGTGGTAGCCATTCTGTTGGATTTGGATAGTG
TCCTTAT
TCTCTGATTTGAACAGTAGATCAGGACGAGTGAGAGGGATGCAGAGGTTGGATTGGAGTGGTTGAGCTATAAAATTTAG
AGGCGC
GCCGTATCGAGTTTTCACATGGAAGTCAAAGCGTACAGTGCGAGCTTGTACGTTGGTCTTAGTATCCCACAAGCTTCTG
TCTAGGT
ATGATGATGGCTATAAGTCACCCAAGGCAGAACTCATCTTGAAGATTGTCTAGAGTGATTTTACCGCTGATGAAATGAC
TGGACT
CCCTCCTCCTGCTCTTATACGAAAAATTGCCTGACTCTGCAAAGGTTGTTTGTCTTGGAAGATGATGTGCCCCCCCATC
GCTCTTAT
CTCATACCCCGCCATCTTTCTAGATTCTCATCTTCAACAAGAGGGGCAATCCATGATCTGCGATCCAGATGTGCTTCTG
GCCTCAT
ACTCTGCCTTCAGGTTGATGTTCACTTAATTGGTGACGAATTCAGCTGATTTGCTGCAGTATGCTTTGTGTTGGTTCTT
TCCAGGCT
TGTGCCAGCCATGAGCGCTTTGAGAGCATGTTGTCACCTATAAACTCGAGTAACGGCCACATATTGTTCACTACTTGAA
TCACATA
CCTAATTTTGATAGAATTGACATGTTTAAAGAGCTGAGGTAGCTTTAATGCCTCTGAAGTATTGTGACACAGCTTCTCA
CAGAGTG
AGAATGAAAAGTTGGACTCCCCCTAATGAAGTAAAAGTTTCGTCTCTGAACGGTGAAGAGCATAGATCCGGCATCAACT
ACCTGG
CTAGACTACGACGTCAATTCTGCGGCCTTTTGACCTTTATATATGTCCATTAATGCAATAGATTCTTTTTTTTTTTTTT
TTTTTTTTTT
TTTTTTTTTTTTTTTTTGCCCAATTTCGCAGATCAAAGTGGACGTTATAGCATCATAACTAAGCTCAGTTGCTGAGGGA
AGCCGTCT
ACTACCTTAGCCCATCCATCCAGCTCCATACCTTGATACTTTAGACGTGAAGCAATTCACACTGTACGTCTCGCAGCTC
TCCTTCCC
GCTCTTGCTTCCCCACTGGGGTCCATGGTGCGTGTATCGTCCCCTCCTTAATTAAGGCCATTTAGGCCGTTGCTGGCGT
TTTTCCAT
AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGAT
ACCAG
GCGTTTCC CCCTGGAAGCTC CCTCGTGC GCTCTCCTGTTCC GAC CCTGC CGCTTACCGGATACCTGTCC
GCCTTTCTCCCTTCGGGA
AGCGTGGCGCTTTCTCATAGCTCAC GCTGTAGGTATCTCAGTTC GGTGTAGGTC GTTC
GCTCCAAGCTGGGCTGTGTGCACGAACC
CCC CGTTCAGCCC GACCGCT GCGCC TTAT CCGGTAACTAT CGTC TT GAGTCC AACC
CGGTAAGACACGACTTATC GCCACTGGCAG
CAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGG
CTACAC
TAGAAGGACAGTATTTGGTATCTGC GCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATC
CGGCAAAC AA
ACCACC GCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATC
CTTT GATC TT
TTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGCCTGCAGGGCCGATTTTGGTCATGAGATTATCAA
AAAGGA
TCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAG
TTACCAA
TGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGA
TAACTACG
ATAC GGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATAC CGCGAGAC CCACGCTCACC
GGCTCCAGATTTATCAGCAATAA
ACCAGC CAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGC CTC
CATCCAGTCTATTAATTGTTGC CGGGAAGC
TAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCG
TTTGGTA
TGGCTTCATTCAGCTCCGGTTC CCAACGATCAAGGC GAGTTACATGATCCC
CCATGTTGTGCAAAAAAGCGGTTAGCTC CTTCGGT
CCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTG
TCATGCC
ATCC GTAAGATGCTTTTCTGTGACTGGTGAGTACTCAAC
CAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCC
CGGCGTCAATACGGGATAATACC GCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAAC
GTTCTTCGGGGCGAAAACT
CTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACT
TTCACCAG
CGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCC GCAAAAAAGGGAATAAGGGC
GACACGGAAATGTTGAATACTCATACT
CTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGGCCATTTAGGCCT (Seq ID no 12)
[00296] SEQ ID NO: 13 = Nucleotide sequence of the pTrex8gM TRI035 expression construct.
CTAGAGTTGTGAAGTC GGTAATC CCGCTGTATAGTAATAC GAGTCGCATCTAAATACTCC
GAAGCTGCTGCGAACCCGGAGAATC
GAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAA
TCATGG
CGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTC CCGAAAAAACTC GGAGATTC
CTAAGTAGCGAT
GGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAAC
TGTTCC
GTAC CCCACCTCTTCTCAACCTTTGGC GTTT CC CTGATTCAGC GTACC
CGTACAAGTCGTAATCACTATTAACCCAGACTGACC GG
ACGTGTTTTGC CCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGAC
CGACTGGGGCTGTTC GAAGCCCGA
ATGTAGGATTGTTATC CGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGC
CTCGAAGGTTCACGGCAA
GGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCC GCAATGCAGCATCACTGGAAAATACAAAC
CAATGGCTAAAA
GTACATAAGTTAATGC CTAAAGAAGTCATATAC
CAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAA
CGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGG
TGATCCC
CCAATTGGGTCGCTTGTTTGTTCC GGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTC
GGAGCGTTTTGCATACAACCAA
GGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAG
GTTTGT
CTGCCGATACGACGAATACTGTATAGTCACTTCTGATGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCA
AAAGAT
TGAGTTGAAAC TGCC TAAGAT CT CGGGCCCT CGGGCC TTC GGCCTTTGGGTGTACATGTTTGTGCTCC
GGGCAAATGCAAAGTGTG
GTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGT
TTCGAA
TAGAAAGAGAAGCTTAGCCAAGAACAATAGC CGATAAAGATAGC CTCATTAAAC
GGAATGAGCTAGTAGGCAAAGTCAGCGAAT
GTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGAC
TTGTACA
CCATCTTTTGAGGCACAGAAACCCAATAGTCAAC CATCACAAGTTTGTACAAAAAAGCAGGCTTCACCATGCAGAC
CTTCGGTGC
TTTTCTCGTTTC CTTCCTC GCCGCCAGGTAAGTTGGCCTTGATGAACCATATCATATATCGCC GAGAAGTGGAC
CGCGTGCTGAGA
CT GAGACAGCGGCCTGGCC GCGGC CAACGCTCCTGGTGGACCTGGTGGTCACGGCCGCAAGCTCC
CCGTCAACCC CAAGACCTTC
CCCAACGAGATCCGCCTCAAGGAC CTCCTCCAC GGCAGCCAGAAGCTCGAAGATTTCGCCTACGC CTACC
CCGAGC GCAACCGCG
TCTTTGGCGGCCAGGCCCAC CTCGACACCGTCAACTACCTCTACCGCGAGCTGAAGAAGAC CGGCTACTACGAC
GTC TACAAGC A
GCCC CAGGTGCACCAGTGGACCC GAGCCGACC AGTCT CTC ACT CTCGGC GGCGACAGCATC CAGGC
CAGCACCATGACCTACAGC
CCCAGCGTCAACGTCACCGCCC CT CTCAGCC TCGT CAGCAAGC T CGGCTGCGC CGAGGGC
GACTACAGCGCCGATGTCAAGGGCA
AGATCGCCCTCGTCAGCCGAGGCGAGTGCAGCTTCGCCCAGAAGTCCGTCCTCAGCGCCAAGGCTGGCGCCGTCGCCAC
CATCGT
CTACAACAACGTCGACGGCAGCCTCGCCGGCACCCTTGGCGGAGCTACTTCTGAGCTGGGCCCCTACTCCCCCATCATC
GGCATC
ACTCTCGCCGCTGGCCAGGACCTCGTCGCCCGACTTCAGGCCGCTCCTACCGAGGTCAGCCTCTGGATCGACAGCAAGG
TCGAGA
ACCGCACCACCTACAACGTCATTGCCCAGACCAAGGGCGGCGACCCCAACAACGTCGTCGCTCTCGGCGGCCACACCGA
CAGCGT
TGAGAACGGCCCTGGCATCAACGACGACGGCTCCGGCGTCATCAGCAACCTCGTCGTCGCCAAGGCCCTCACCCGCTAC
AGCGTC
AAGAACGCCGTCCGCTTCTGCTTCTGGACCGCCGAAGAGTTCGGCCTCCTCGGCAGCAACTACTACGTCGACAACCTCA
GCCCTG
CCGAGCTGGCCAAGATCCGCCTCTACCTCAACTTCGACATGATCGCCAGCCCCAACTACGCCCTCATGATCTACGACGG
CGACGG
CAGCGCCTTCAACCTCACTGGACCCCCTGGCAGCGCCCAGATCGAGAGCCTCTTCGAGAACTACTACAAGAGCATCAAG
CAGGGC
TTCGTCCCCACCGCCTTCGACGGCCGATCTGACTACGAGGGCTTCATCCTCAAGGGCATCCCCGCTGGCGGCGTCTTTA
CTGGCGC
CGAGAGCCTCAAGACCGAGGAACAGGCCCGCCTGTTCGGCGGCCAGGCTGGCGTTGCTCTCGACGCCAACTACCACGCC
AAGGG
CGACAACATGACCAACCTCAACCACAAGGCCTTTCTCATCAACAGCCGCGCCACGGCCTTCGCCGTCGCTACCTACGCC
AACAAC
CTCAGCAGCATCCCCCCTCGCAACGCCACCGTCGTCAAGCGCGAGAGCATGAAGTGGACCAAGCGCGAGGAACCCCACA
CCCAC
GGCGCCGACACTGGCTGCTTTGCCAGCCGCGTCAAGGAGTAAGACCCAGCTTTCTTGTACAAAGTGGTGATCGCGCCAG
CTCCGT
GCGAAAGCCTGACGCACCGGTAGATTCTTGGTGAGCCCGTATCATGACGGCGGCGGGAGCTACATGGCCCCGGGTGATT
TATTTT
TTTTGTATCTACTTCTGACCCTTTTCAAATATACGGTCAACTCATCTTTCACTGGAGATGCGGCCTGCTTGGTATTGCG
ATGTTGTC
AGCTTGGCAAATTGTGGCTTTCGAAAACACAAAACGATTCCTTAGTAGCCATGCATTTTAAGATAACGGAATAGAAGAA
AGAGGA
AATTAAAAAAAAAAAAAAAACAAACATCCCGTTCATAACCCGTAGAATCGCCGCTCTTCGGCTAGCTAGTTACGCTTGT
TTATTTA
CGACAAGATCTAGAAGATTCGAGATAGAATAATAATAATAACAACAATTTGCCTCTTCTTTCCACCTTTTCAGTCTTAC
TCTCCCTT
CTGACATTGAACGCCTCAATCAGTCAGTCGCCTTGTACTTGGCACGGTAATCCTCCGTGTTCTTGATATCCTCAGGGGT
AGCAAAG
CCCTTCATGCCATCGATAATGTCATCCAGAGTGAGGATGGCAAAGATGGGGATGCCGTACTCCTTCCTCAGCTCGCCAA
TGGCAC
TCGGTCCAGGCTTGGAGTCGTCGCCATCCGCAGCGGGGAGCTTCTCCATGCGGTCCAGGGCCACGACGATGCCGGCGAC
GATGCC
GCCCTCCTTGGTGATCTTCTCAATGGCGTCCCTCTTGGCGGTGCCGGCGGTGATGACGTCGTCGACAATCAGGACCCTC
TTGCCCT
TGAGCGAAGCGCCGACGATGTTGCCGCCCTCGCCGTGGTCCTTGGCCTCCTTGCGGTCAAACGAGTAGGAGACGCGGTC
CAGGTT
CTGGGGCGCCAGCTCGCCGAGCTTGATGGTGATGGCGGAGCACAGCGGGATGCCCTTGTAGGCCGGGCCGAAGACGATG
TCGAA
CTCTAGGCCGGCCTTCTCCTGGGCCTCGATGATGGTCTTTGCAAAGGCGGAGGCGATGGCGCCGGCGAGGCGCGCCGTG
TGGAAT
TCGCCCGCGTTGAAGAAGTAGGGGGATATCCGCTTGGACTTGAGCTCGAAGCTGCCAAACTTGAGGACGCCGCCGTCGA
TGGCGG
ATTTGAGGAAGTCCTGCTTGTAGGCAGGCAGCTGGGAGGTGGTAGCCATTCTGTTGGATTTGGATAGTGTCCTTATTCT
CTGATTT
GAACAGTAGATCAGGACGAGTGAGAGGGATGCAGAGGTTGGATTGGAGTGGTTGAGCTATAAAATTTAGAGGCGCGCCG
TATCG
AGTTTTCACATGGAAGTCAAAGCGTACAGTGCGAGCTTGTACGTTGGTCTTAGTATCCCACAAGCTTCTGTCTAGGTAT
GATGATG
GCTATAAGTCACCCAAGGCAGAACTCATCTTGAAGATTGTCTAGAGTGATTTTACCGCTGATGAAATGACTGGACTCCC
TCCTCCT
GCTCTTATACGAAAAATTGCCTGACTCTGCAAAGGTTGTTTGTCTTGGAAGATGATGTGCCCCCCCATCGCTCTTATCT
CATACCCC
GCCATCTTTCTAGATTCTCATCTTCAACAAGAGGGGCAATCCATGATCTGCGATCCAGATGTGCTTCTGGCCTCATACT
CTGCCTTC
AGGTTGATGTTCACTTAATTGGTGACGAATTCAGCTGATTTGCTGCAGTATGCTTTGTGTTGGTTCTTTCCAGGCTTGT
GCCAGCCA
TGAGCGCTTTGAGAGCATGTTGTCACCTATAAACTCGAGTAACGGCCACATATTGTTCACTACTTGAATCACATACCTA
ATTTTGA
TAGAATTGACATGTTTAAAGAGCTGAGGTAGCTTTAATGCCTCTGAAGTATTGTGACACAGCTTCTCACAGAGTGAGAA
TGAAAA
GTTGGACTCCCCCTAATGAAGTAAAAGTTTCGTCTCTGAACGGTGAAGAGCATAGATCCGGCATCAACTACCTGGCTAG
ACTACG
ACGTCAATTCTGCGGCCTTTTGACCTTTATATATGTCCATTAATGCAATAGATTCTTTTTTTTTTTTTTTTTTTTTTTT
TTTTTTTTTTT
TTTTTTGCCCAATTTCGCAGATCAAAGTGGACGTTATAGCATCATAACTAAGCTCAGTTGCTGAGGGAAGCCGTCTACT
ACCTTAG
CCCATCCATCCAGCTCCATACCTTGATACTTTAGACGTGAAGCAATTCACACTGTACGTCTCGCAGCTCTCCTTCCCGC
TCTTGCTT
CCCCACTGGGGTCCATGGTGCGTGTATCGTCCCCTCCTTAATTAAGGCCATTTAGGCCGTTGCTGGCGTTTTTCCATAG
GCTCCGCC
CCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTT
TCCCC
CTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAG
CGTGGCGC
TTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCC
CGTTCAG
CCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAG
CCACTG
GTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAG
AAGGA
CAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAAC
CACCGCT
GGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTT
CTACGGG
GTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGCCTGCAGGGCCGATTTTGGTCATGAGATTATCAAAAAGGATCT
TCACCT
AGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATG
CTTAATC
AGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATC CATAGTTGCCTGACTCCCCGTCGTGTAGATAACTAC
GATAC GGGA
GGGCTTACCATCTGGCCCCAGTGCTGCAATGATACC GCGAGACC CACGCTCACC
GGCTCCAGATTTATCAGCAATAAACCAGCCA
GCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTA
GAGTAA
GTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTAT
GGCTTCAT
TCAGCTCCGGTTC CCAACGATCAAGGC GAGTTACATGATCCCC CAT GTTGT GCAAAAAAGC
GGTTAGCTCCTTC GGTCCTCCGATC
GTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCAT
CCGTAAG
ATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCG
GCGTCAA
TACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTC
AAGGAT
CTTACCGCTGTTGAGATCCAGTTCGATGTAACC CACTCGTGCAC
CCAACTGATCTTCAGCATCTTTTACTTTCACCAGC GTTTCTGG
GTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTC
CTTTT
TCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGGCCATTTAGGCCT (Seq ID no 13)
CTAGAGTTGTGAAGTC GGTAATC CCGCTGTATAGTAATAC GAGTCGCATCTAAATACTCC
GAAGCTGCTGCGAACCCGGAGAATC
GAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAA
TCATGG
CGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTC CCGAAAAAACTC GGAGATTC
CTAAGTAGCGAT
GGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAAC
TGTTCC
GTAC CCCACCTCTTCTCAACCTTTGGC GTTT CC CTGATTCAGC GTACC
CGTACAAGTCGTAATCACTATTAACCCAGACTGACC GG
ACGTGTTTTGC CCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGAC
CGACTGGGGCTGTTC GAAGCCCGA
ATGTAGGATTGTTATC CGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGC
CTCGAAGGTTCACGGCAA
GGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCC GCAATGCAGCATCACTGGAAAATACAAAC
CAATGGCTAAAA
GTACATAAGTTAATGC CTAAAGAAGTCATATAC
CAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAA
CGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGG
TGATCCC
CCAATTGGGTCGCTTGTTTGTTCC GGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTC
GGAGCGTTTTGCATACAACCAA
GGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAG
GTTTGT
CTGCCGATACGACGAATACTGTATAGTCACTTCTGATGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCA
AAAGAT
TGAGTTGAAAC TGCC TAAGAT CT CGGGCCCT CGGGCC TTC GGCCTTTGGGTGTACATGTTTGTGCTCC
GGGCAAATGCAAAGTGTG
GTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGT
TTCGAA
TAGAAAGAGAAGCTTAGCCAAGAACAATAGC CGATAAAGATAGC CTCATTAAAC
GGAATGAGCTAGTAGGCAAAGTCAGCGAAT
GTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGAC
TTGTACA
CCATCTTTTGAGGCACAGAAACCCAATAGTCAAC CATCACAAGTTTGTACAAAAAAGCAGGCTTCACCATGCAGAC
CTTCGGTGC
TTTTCTCGTTTC CTTCCTC GCCGCCAGGTAAGTTGGCCTTGATGAACCATATCATATATCGCC GAGAAGTGGAC
CGCGTGCTGAGA
CT GAGACAGCGGCCTGGCC GCGGC CAACGCTCCTGGTGGACCTGGTGGTCACGGCCGCAAGCTCC
CCGTCAACCC CAAGACCTTC
CCCAACGAGATCCGCCTCAAGGAC CTCCTCCAC GGCAGCCAGAAGCTCGAAGATTTCGCCTACGC CTACC
CCGAGC GCAACCGCG
TCTTTGGCGGCCAGGCCCAC CTCGACACCGTCAACTACCTCTACCGCGAGCTGAAGAAGAC CGGCTACTACGAC
GTC TACAAGC A
GCCC CAGGTGCACCAGTGGACCC GAGCCGACC AGTCT CTC ACT CTCGGC GGCGACAGCATC CAGGC
CAGCACCATGACCTACAGC
CCCAGCGTCAACGTCACCGCCC CT CTCAGCC TCGT CAGCAAGC T CGGCTGCGC CGAGGGC
GACTACAGCGCCGATGTCAAGGGCA
AGATCGCCCTCGTCAGCCGAGGCGAGTGCAGCTTCGCCCAGAAGTCCGTCCTCAGCGCCAAGGCTGGCGCCGTCGCCAC
CATCGT
CTACAACAACGTCGACGGCAGCCTCGCCGGCACCCTTGGCGGAGCTACTTCTGAGCTGGGCCCCTACTCCCCCATCATC
GGCATC
ACTCTCGCCGCTGGCCAGGACCTCGTCGCCCGACTTCAGGCCGCTCCTACCGAGGTCAGCCTCTGGATCGACAGCAAGG
TCGAGA
ACCGCACCACCTACAACGTCATTGCCCAGACCAAGGGCGGCGACCCCAACAACGTCGTCGCTCTCGGCGGCCACACCGA
CAGCGT
TGAGAACGGCCCTGGCATCAACGACGACGGCTCCGGCGTCATCAGCAACCTCGTCGTCGCCAAGGCCCTCACCCGCTAC
AGCGTC
AAGAACGCCGTCCGCTTCTGCTTCTGGACCGCCGAAGAGTTCGGCCTCCTCGGCAGCAACTACTACGTCGACAACCTCA
GCCCTG
CCGAGCTGGCCAAGATCCGCCTCTACCTCAACTTCGACATGATCGCCAGCCCCAACTACGCCCTCATGATCTACGACGG
CGACGG
CAGCGCCTTCAACCTCACTGGACCCCCTGGCAGCGCCCAGATCGAGAGCCTCTTCGAGAACTACTACAAGAGCATCAAG
CAGGGC
TTCGTCCCCACCGCCTTCGACGGCCGATCTGACTACGAGGGCTTCATCCTCAAGGGCATCCCCGCTGGCGGCGTCTTTA
CTGGCGC
CGAGAGCCTCAAGACCGAGGAACAGGCCCGCCTGTTCGGCGGCCAGGCTGGCGTTGCTCTCGACGCCAACTACCACGCC
AAGGG
CGACAACATGACCAACCTCAACCACAAGGCCTTTCTCATCAACAGCCGCGCCACGGCCTTCGCCGTCGCTACCTACGCC
AACAAC
CTCAGCAGCATCCCCCCTCGCAACGCCACCGTCGTCAAGCGCGAGAGCATGAAGTGGACCAAGCGCGAGGAACCCCACA
CCCAC
GGCGCCGACACTGGCTGCTTTGCCAGCCGCGTCAAGGAGTAAGACCCAGCTTTCTTGTACAAAGTGGTGATCGCGCCAG
CTCCGT
GCGAAAGCCTGACGCACCGGTAGATTCTTGGTGAGCCCGTATCATGACGGCGGCGGGAGCTACATGGCCCCGGGTGATT
TATTTT
TTTTGTATCTACTTCTGACCCTTTTCAAATATACGGTCAACTCATCTTTCACTGGAGATGCGGCCTGCTTGGTATTGCG
ATGTTGTC
AGCTTGGCAAATTGTGGCTTTCGAAAACACAAAACGATTCCTTAGTAGCCATGCATTTTAAGATAACGGAATAGAAGAA
AGAGGA
AATTAAAAAAAAAAAAAAAACAAACATCCCGTTCATAACCCGTAGAATCGCCGCTCTTCGGCTAGCTAGTTACGCTTGT
TTATTTA
CGACAAGATCTAGAAGATTCGAGATAGAATAATAATAATAACAACAATTTGCCTCTTCTTTCCACCTTTTCAGTCTTAC
TCTCCCTT
CTGACATTGAACGCCTCAATCAGTCAGTCGCCTTGTACTTGGCACGGTAATCCTCCGTGTTCTTGATATCCTCAGGGGT
AGCAAAG
CCCTTCATGCCATCGATAATGTCATCCAGAGTGAGGATGGCAAAGATGGGGATGCCGTACTCCTTCCTCAGCTCGCCAA
TGGCAC
TCGGTCCAGGCTTGGAGTCGTCGCCATCCGCAGCGGGGAGCTTCTCCATGCGGTCCAGGGCCACGACGATGCCGGCGAC
GATGCC
GCCCTCCTTGGTGATCTTCTCAATGGCGTCCCTCTTGGCGGTGCCGGCGGTGATGACGTCGTCGACAATCAGGACCCTC
TTGCCCT
TGAGCGAAGCGCCGACGATGTTGCCGCCCTCGCCGTGGTCCTTGGCCTCCTTGCGGTCAAACGAGTAGGAGACGCGGTC
CAGGTT
CTGGGGCGCCAGCTCGCCGAGCTTGATGGTGATGGCGGAGCACAGCGGGATGCCCTTGTAGGCCGGGCCGAAGACGATG
TCGAA
CTCTAGGCCGGCCTTCTCCTGGGCCTCGATGATGGTCTTTGCAAAGGCGGAGGCGATGGCGCCGGCGAGGCGCGCCGTG
TGGAAT
TCGCCCGCGTTGAAGAAGTAGGGGGATATCCGCTTGGACTTGAGCTCGAAGCTGCCAAACTTGAGGACGCCGCCGTCGA
TGGCGG
ATTTGAGGAAGTCCTGCTTGTAGGCAGGCAGCTGGGAGGTGGTAGCCATTCTGTTGGATTTGGATAGTGTCCTTATTCT
CTGATTT
GAACAGTAGATCAGGACGAGTGAGAGGGATGCAGAGGTTGGATTGGAGTGGTTGAGCTATAAAATTTAGAGGCGCGCCG
TATCG
AGTTTTCACATGGAAGTCAAAGCGTACAGTGCGAGCTTGTACGTTGGTCTTAGTATCCCACAAGCTTCTGTCTAGGTAT
GATGATG
GCTATAAGTCACCCAAGGCAGAACTCATCTTGAAGATTGTCTAGAGTGATTTTACCGCTGATGAAATGACTGGACTCCC
TCCTCCT
GCTCTTATACGAAAAATTGCCTGACTCTGCAAAGGTTGTTTGTCTTGGAAGATGATGTGCCCCCCCATCGCTCTTATCT
CATACCCC
GCCATCTTTCTAGATTCTCATCTTCAACAAGAGGGGCAATCCATGATCTGCGATCCAGATGTGCTTCTGGCCTCATACT
CTGCCTTC
AGGTTGATGTTCACTTAATTGGTGACGAATTCAGCTGATTTGCTGCAGTATGCTTTGTGTTGGTTCTTTCCAGGCTTGT
GCCAGCCA
TGAGCGCTTTGAGAGCATGTTGTCACCTATAAACTCGAGTAACGGCCACATATTGTTCACTACTTGAATCACATACCTA
ATTTTGA
TAGAATTGACATGTTTAAAGAGCTGAGGTAGCTTTAATGCCTCTGAAGTATTGTGACACAGCTTCTCACAGAGTGAGAA
TGAAAA
GTTGGACTCCCCCTAATGAAGTAAAAGTTTCGTCTCTGAACGGTGAAGAGCATAGATCCGGCATCAACTACCTGGCTAG
ACTACG
ACGTCAATTCTGCGGCCTTTTGACCTTTATATATGTCCATTAATGCAATAGATTCTTTTTTTTTTTTTTTTTTTTTTTT
TTTTTTTTTTT
TTTTTTGCCCAATTTCGCAGATCAAAGTGGACGTTATAGCATCATAACTAAGCTCAGTTGCTGAGGGAAGCCGTCTACT
ACCTTAG
CCCATCCATCCAGCTCCATACCTTGATACTTTAGACGTGAAGCAATTCACACTGTACGTCTCGCAGCTCTCCTTCCCGC
TCTTGCTT
CCCCACTGGGGTCCATGGTGCGTGTATCGTCCCCTCCTTAATTAAGGCCATTTAGGCCGTTGCTGGCGTTTTTCCATAG
GCTCCGCC
CCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTT
TCCCC
CTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAG
CGTGGCGC
TTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCC
CGTTCAG
CCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAG
CCACTG
GTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAG
AAGGA
CAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAAC
CACCGCT
GGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTT
CTACGGG
GTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGCCTGCAGGGCCGATTTTGGTCATGAGATTATCAAAAAGGATCT
TCACCT
AGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATG
CTTAATC
AGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATC CATAGTTGCCTGACTCCCCGTCGTGTAGATAACTAC
GATAC GGGA
GGGCTTACCATCTGGCCCCAGTGCTGCAATGATACC GCGAGACC CACGCTCACC
GGCTCCAGATTTATCAGCAATAAACCAGCCA
GCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTA
GAGTAA
GTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTAT
GGCTTCAT
TCAGCTCCGGTTC CCAACGATCAAGGC GAGTTACATGATCCCC CAT GTTGT GCAAAAAAGC
GGTTAGCTCCTTC GGTCCTCCGATC
GTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCAT
CCGTAAG
ATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCG
GCGTCAA
TACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTC
AAGGAT
CTTACCGCTGTTGAGATCCAGTTCGATGTAACC CACTCGTGCAC
CCAACTGATCTTCAGCATCTTTTACTTTCACCAGC GTTTCTGG
GTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTC
CTTTT
TCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGGCCATTTAGGCCT (Seq ID no 13)
[00297] SEQ ID NO: 14 = Nucleotide sequence of the pTrex8gM TRI036 expression construct.
CTAGAGTTGTGAAGTC GGTAATC CCGCTGTATAGTAATAC GAGTCGCATCTAAATACTCC
GAAGCTGCTGCGAACCCGGAGAATC
GAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAA
TCATGG
CGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTC CCGAAAAAACTC GGAGATTC
CTAAGTAGCGAT
GGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAAC
TGTTCC
GTAC CCCACCTCTTCTCAACCTTTGGC GTTT CC CTGATTCAGC GTACC
CGTACAAGTCGTAATCACTATTAACCCAGACTGACC GG
ACGTGTTTTGC CCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGAC
CGACTGGGGCTGTTC GAAGCCCGA
ATGTAGGATTGTTATC CGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGC
CTCGAAGGTTCACGGCAA
GGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCC GCAATGCAGCATCACTGGAAAATACAAAC
CAATGGCTAAAA
GTACATAAGTTAATGC CTAAAGAAGTCATATAC
CAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAA
CGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGG
TGATCCC
CCAATTGGGTCGCTTGTTTGTTCC GGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTC
GGAGCGTTTTGCATACAACCAA
GGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAG
GTTTGT
CTGCCGATACGACGAATACTGTATAGTCACTTCTGATGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCA
AAAGAT
TGAGTTGAAAC TGCC TAAGAT CT CGGGCCCT CGGGCC TTC GGCCTTTGGGTGTACATGTTTGTGCTCC
GGGCAAATGCAAAGTGTG
GTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGT
TTCGAA
TAGAAAGAGAAGCTTAGCCAAGAACAATAGC CGATAAAGATAGC CTCATTAAAC
GGAATGAGCTAGTAGGCAAAGTCAGCGAAT
GTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGAC
TTGTACA
CCATCTTTTGAGGCACAGAAACCCAATAGTCAAC CATCACAAGTTTGTACAAAAAAGCAGGCTTCACCATGCAGAC
CTTCGGTGC
TTTTCTCGTTTC CTTCCTC GCCGCCAGGTAAGTTGGCCTTGATGAACCATATCATATATCGCC GAGAAGTGGAC
CGCGTGCTGAGA
CT GAGACAGCGGC CTGGCC GCGGC CGGTGGC CCTCATGGATTTGGCCTCCCCAAGATCGAC CT CCGCCC
TATGGT CAGCAGC AAC
CGCCTCCAGAGCATGATCACC CTCAAGGACCTCATGGACGGCGC
CAAGAAGCTCCAGGACATTGCCACCAAGAACGGCGGCAAC
CGCGC CTTTGGCGGC GCTGGC CACAACGC CACTGTCGACTAC CT CTACAAGACCCT CACCAGC CTC
GGCGGCTACTAC ACCGTC A
AGAAGCAGCCCTTCAAGGAAATCTTCAGCAGCGGCAGCGGCAGC CTCATCGTC GACGGCCAGGGCATCGACGCC
GGCATCATGA
CCTATACC CCTGGCGGCAGCGCCACC GCCAAC CTCGTCCAGGTTGCTAACCTCGGCTGCGAGGAC GAGGACTAC
CCTGCCGAGGT
CGCC GGCAACATTGCCCTCATTAGCC
GCGGCAGCTGCACCTTCAGCAGCAAGAGCCTCAAGGCCAAGGCCGCTGGCGC CGTCGGC
GCTATCGTCTACAACAACGTCCC CGGCGAGCTGAGCGGAACCCTCGGCACCCCCTTTCTCGACTAC GCCCC
CATCGTCGGCATCAG
CCAAGAGGACGGCCAGGTCATCCTTGAGAAGCTCGCC GCTGGC CCCGTCACC GCCAC
CCTCAACATCGACGCCATCGTCGAGGAA
CGCAC CACCTACAACGTCATTGCCGAGACTAAGGAAGGCGACCACAACAACGTGCTCATTGTC
GGCGGCCACAGCGACAGCGTT
GCTGCCGGCCCTGGCATCAACGACGACGGCTCTGGCACCATCGGCATCCTCACCGTCGCCAAGGCCCTCGCCAAGGCCA
ACGTCC
GCATCAAGAACGCCGTCCGCTTCGCCTTCTGGTCCGCCGAAGAGTTCGGCCTCCTCGGCAGCTACGCCTACATGAAGTC
CCTCAAC
GAGAGCGAGGCCGAGGTGGCCAAGATCCGCGCCTACCTCAACTTCGACATGATCGCCAGCCCCAACTACATCTACGGCA
TCTACG
ACGGCGACGGCAACGCCTTCAACCTCACTGGCCCTGCCGGCAGCGACATCATCGAGAAGGACTTCGAGGACTTCTTCAA
GAAGAA
GAAGACCCCCAGCGTCCCCACCGAGTTCAGCGGCCGATCTGACTACGCCGCCTTCATCGAGAACGGCATCCCCAGCGGC
GGACTC
TTCACTGGCGCCGAGGTCCTCAAGACCGAGGAAGAGGCCAAGCTGTTCGGCGGCAAGGCCGGCGTCGCCTACGACGTCA
ACTAC
CACAAGGCCGGCGACACCGTCGACAACCTCGCCAAGGACGCCTTCCTGCTCAACACCAAGGCCATTGCCAACAGCGTCG
CCAAGT
ACGCCGCCAGCTGGGCCGGCTTTCCTAAGCCTTCTGCCGTCCGCCGACGCTACGACGCCGATATGGCCCAGCTCCTCAA
GCGCTCT
GGCGGCGTTCATGGCCACGGCCCTCACACTCATAGCGGCCCTTGTGGCGGCGGTGACCTCCTCTAAGACCCAGCTTTCT
TGTACAA
AGTGGTGATCGCGCCAGCTCCGTGCGAAAGCCTGACGCACCGGTAGATTCTTGGTGAGCCCGTATCATGACGGCGGCGG
GAGCTA
CATGGCCCCGGGTGATTTATTTTTTTTGTATCTACTTCTGACCCTTTTCAAATATACGGTCAACTCATCTTTCACTGGA
GATGCGGC
CTGCTTGGTATTGCGATGTTGTCAGCTTGGCAAATTGTGGCTTTCGAAAACACAAAACGATTCCTTAGTAGCCATGCAT
TTTAAGA
TAACGGAATAGAAGAAAGAGGAAATTAAAAAAAAAAAAAAAACAAACATCCCGTTCATAACCCGTAGAATCGCCGCTCT
TCGGC
TAGCTAGTTACGCTTGTTTATTTACGACAAGATCTAGAAGATTCGAGATAGAATAATAATAATAACAACAATTTGCCTC
TTCTTTC
CACCTTTTCAGTCTTACTCTCCCTTCTGACATTGAACGCCTCAATCAGTCAGTCGCCTTGTACTTGGCACGGTAATCCT
CCGTGTTC
TTGATATCCTCAGGGGTAGCAAAGCCCTTCATGCCATCGATAATGTCATCCAGAGTGAGGATGGCAAAGATGGGGATGC
CGTACT
CCTTCCTCAGCTCGCCAATGGCACTCGGTCCAGGCTTGGAGTCGTCGCCATCCGCAGCGGGGAGCTTCTCCATGCGGTC
CAGGGCC
ACGACGATGCCGGCGACGATGCCGCCCTCCTTGGTGATCTTCTCAATGGCGTCCCTCTTGGCGGTGCCGGCGGTGATGA
CGTCGTC
GACAATCAGGACCCTCTTGCCCTTGAGCGAAGCGCCGACGATGTTGCCGCCCTCGCCGTGGTCCTTGGCCTCCTTGCGG
TCAAACG
AGTAGGAGACGCGGTCCAGGTTCTGGGGCGCCAGCTCGCCGAGCTTGATGGTGATGGCGGAGCACAGCGGGATGCCCTT
GTAGG
CCGGGCCGAAGACGATGTCGAACTCTAGGCCGGCCTTCTCCTGGGCCTCGATGATGGTCTTTGCAAAGGCGGAGGCGAT
GGCGCC
GGCGAGGCGCGCCGTGTGGAATTCGCCCGCGTTGAAGAAGTAGGGGGATATCCGCTTGGACTTGAGCTCGAAGCTGCCA
AACTTG
AGGACGCCGCCGTCGATGGCGGATTTGAGGAAGTCCTGCTTGTAGGCAGGCAGCTGGGAGGTGGTAGCCATTCTGTTGG
ATTTGG
ATAGTGTCCTTATTCTCTGATTTGAACAGTAGATCAGGACGAGTGAGAGGGATGCAGAGGTTGGATTGGAGTGGTTGAG
CTATAA
AATTTAGAGGCGCGCCGTATCGAGTTTTCACATGGAAGTCAAAGCGTACAGTGCGAGCTTGTACGTTGGTCTTAGTATC
CCACAA
GCTTCTGTCTAGGTATGATGATGGCTATAAGTCACCCAAGGCAGAACTCATCTTGAAGATTGTCTAGAGTGATTTTACC
GCTGATG
AAATGACTGGACTCCCTCCTCCTGCTCTTATACGAAAAATTGCCTGACTCTGCAAAGGTTGTTTGTCTTGGAAGATGAT
GTGCCCC
CCCATCGCTCTTATCTCATACCCCGCCATCTTTCTAGATTCTCATCTTCAACAAGAGGGGCAATCCATGATCTGCGATC
CAGATGT
GCTTCTGGCCTCATACTCTGCCTTCAGGTTGATGTTCACTTAATTGGTGACGAATTCAGCTGATTTGCTGCAGTATGCT
TTGTGTTG
GTTCTTTCCAGGCTTGTGCCAGCCATGAGCGCTTTGAGAGCATGTTGTCACCTATAAACTCGAGTAACGGCCACATATT
GTTCACT
ACTTGAATCACATACCTAATTTTGATAGAATTGACATGTTTAAAGAGCTGAGGTAGCTTTAATGCCTCTGAAGTATTGT
GACACAG
CTTCTCACAGAGTGAGAATGAAAAGTTGGACTCCCCCTAATGAAGTAAAAGTTTCGTCTCTGAACGGTGAAGAGCATAG
ATCCGG
CATCAACTACCTGGCTAGACTACGACGTCAATTCTGCGGCCTTTTGACCTTTATATATGTCCATTAATGCAATAGATTC
TTTTTTTT
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCCCAATTTCGCAGATCAAAGTGGACGTTATAGCATCATAACTAAG
CTCAGTTGCT
GAGGGAAGCCGTCTACTACCTTAGCCCATCCATCCAGCTCCATACCTTGATACTTTAGACGTGAAGCAATTCACACTGT
ACGTCTC
GCAGCTCTCCTTCCCGCTCTTGCTTCCCCACTGGGGTCCATGGTGCGTGTATCGTCCCCTCCTTAATTAAGGCCATTTA
GGCCGTTG
CTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA
CAGGAC
TATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCT
GTCCGCC
TTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCA
AGCTGGGC
TGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGAC
ACGACTT
ATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGG
TGGCCT
AACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTA
GCTCTT
GATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATC
TCAAGA
AGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGCCTGCAGGGCCGATTTTGG
TCATGAG
ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAA
ACTTGGT
CTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACT
CCCCGTCG
TGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGC
TCCAGA
TTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCT
ATTAAT
TGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGG
TGTCACG
CT CGTCGTTTGGTATGGC TTCATTC AGCTC CGGTTCCCAAC GATCAAGGCGAGTTACATGATCCC
CCATGTTGTGCAAAAAAGCGG
TTAGCTC CTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCC
GCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTC
TTACTGTCATGCCATC
CGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGC GACCG
AGTTGCTCTTGCCCGGCGTCAATAC GGGATAATACCGCGC
CACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTC
GGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACC CACTC GTGCAC
CCAACTGATCTTCAGCATCTT
TTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA
ATGTT
GAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGGCCATTTAGGCCT
(Seq ID no 14)
CTAGAGTTGTGAAGTC GGTAATC CCGCTGTATAGTAATAC GAGTCGCATCTAAATACTCC
GAAGCTGCTGCGAACCCGGAGAATC
GAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAA
TCATGG
CGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTC CCGAAAAAACTC GGAGATTC
CTAAGTAGCGAT
GGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAAC
TGTTCC
GTAC CCCACCTCTTCTCAACCTTTGGC GTTT CC CTGATTCAGC GTACC
CGTACAAGTCGTAATCACTATTAACCCAGACTGACC GG
ACGTGTTTTGC CCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGAC
CGACTGGGGCTGTTC GAAGCCCGA
ATGTAGGATTGTTATC CGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGC
CTCGAAGGTTCACGGCAA
GGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCC GCAATGCAGCATCACTGGAAAATACAAAC
CAATGGCTAAAA
GTACATAAGTTAATGC CTAAAGAAGTCATATAC
CAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAA
CGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGG
TGATCCC
CCAATTGGGTCGCTTGTTTGTTCC GGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTC
GGAGCGTTTTGCATACAACCAA
GGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAG
GTTTGT
CTGCCGATACGACGAATACTGTATAGTCACTTCTGATGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCA
AAAGAT
TGAGTTGAAAC TGCC TAAGAT CT CGGGCCCT CGGGCC TTC GGCCTTTGGGTGTACATGTTTGTGCTCC
GGGCAAATGCAAAGTGTG
GTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGT
TTCGAA
TAGAAAGAGAAGCTTAGCCAAGAACAATAGC CGATAAAGATAGC CTCATTAAAC
GGAATGAGCTAGTAGGCAAAGTCAGCGAAT
GTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGAC
TTGTACA
CCATCTTTTGAGGCACAGAAACCCAATAGTCAAC CATCACAAGTTTGTACAAAAAAGCAGGCTTCACCATGCAGAC
CTTCGGTGC
TTTTCTCGTTTC CTTCCTC GCCGCCAGGTAAGTTGGCCTTGATGAACCATATCATATATCGCC GAGAAGTGGAC
CGCGTGCTGAGA
CT GAGACAGCGGC CTGGCC GCGGC CGGTGGC CCTCATGGATTTGGCCTCCCCAAGATCGAC CT CCGCCC
TATGGT CAGCAGC AAC
CGCCTCCAGAGCATGATCACC CTCAAGGACCTCATGGACGGCGC
CAAGAAGCTCCAGGACATTGCCACCAAGAACGGCGGCAAC
CGCGC CTTTGGCGGC GCTGGC CACAACGC CACTGTCGACTAC CT CTACAAGACCCT CACCAGC CTC
GGCGGCTACTAC ACCGTC A
AGAAGCAGCCCTTCAAGGAAATCTTCAGCAGCGGCAGCGGCAGC CTCATCGTC GACGGCCAGGGCATCGACGCC
GGCATCATGA
CCTATACC CCTGGCGGCAGCGCCACC GCCAAC CTCGTCCAGGTTGCTAACCTCGGCTGCGAGGAC GAGGACTAC
CCTGCCGAGGT
CGCC GGCAACATTGCCCTCATTAGCC
GCGGCAGCTGCACCTTCAGCAGCAAGAGCCTCAAGGCCAAGGCCGCTGGCGC CGTCGGC
GCTATCGTCTACAACAACGTCCC CGGCGAGCTGAGCGGAACCCTCGGCACCCCCTTTCTCGACTAC GCCCC
CATCGTCGGCATCAG
CCAAGAGGACGGCCAGGTCATCCTTGAGAAGCTCGCC GCTGGC CCCGTCACC GCCAC
CCTCAACATCGACGCCATCGTCGAGGAA
CGCAC CACCTACAACGTCATTGCCGAGACTAAGGAAGGCGACCACAACAACGTGCTCATTGTC
GGCGGCCACAGCGACAGCGTT
GCTGCCGGCCCTGGCATCAACGACGACGGCTCTGGCACCATCGGCATCCTCACCGTCGCCAAGGCCCTCGCCAAGGCCA
ACGTCC
GCATCAAGAACGCCGTCCGCTTCGCCTTCTGGTCCGCCGAAGAGTTCGGCCTCCTCGGCAGCTACGCCTACATGAAGTC
CCTCAAC
GAGAGCGAGGCCGAGGTGGCCAAGATCCGCGCCTACCTCAACTTCGACATGATCGCCAGCCCCAACTACATCTACGGCA
TCTACG
ACGGCGACGGCAACGCCTTCAACCTCACTGGCCCTGCCGGCAGCGACATCATCGAGAAGGACTTCGAGGACTTCTTCAA
GAAGAA
GAAGACCCCCAGCGTCCCCACCGAGTTCAGCGGCCGATCTGACTACGCCGCCTTCATCGAGAACGGCATCCCCAGCGGC
GGACTC
TTCACTGGCGCCGAGGTCCTCAAGACCGAGGAAGAGGCCAAGCTGTTCGGCGGCAAGGCCGGCGTCGCCTACGACGTCA
ACTAC
CACAAGGCCGGCGACACCGTCGACAACCTCGCCAAGGACGCCTTCCTGCTCAACACCAAGGCCATTGCCAACAGCGTCG
CCAAGT
ACGCCGCCAGCTGGGCCGGCTTTCCTAAGCCTTCTGCCGTCCGCCGACGCTACGACGCCGATATGGCCCAGCTCCTCAA
GCGCTCT
GGCGGCGTTCATGGCCACGGCCCTCACACTCATAGCGGCCCTTGTGGCGGCGGTGACCTCCTCTAAGACCCAGCTTTCT
TGTACAA
AGTGGTGATCGCGCCAGCTCCGTGCGAAAGCCTGACGCACCGGTAGATTCTTGGTGAGCCCGTATCATGACGGCGGCGG
GAGCTA
CATGGCCCCGGGTGATTTATTTTTTTTGTATCTACTTCTGACCCTTTTCAAATATACGGTCAACTCATCTTTCACTGGA
GATGCGGC
CTGCTTGGTATTGCGATGTTGTCAGCTTGGCAAATTGTGGCTTTCGAAAACACAAAACGATTCCTTAGTAGCCATGCAT
TTTAAGA
TAACGGAATAGAAGAAAGAGGAAATTAAAAAAAAAAAAAAAACAAACATCCCGTTCATAACCCGTAGAATCGCCGCTCT
TCGGC
TAGCTAGTTACGCTTGTTTATTTACGACAAGATCTAGAAGATTCGAGATAGAATAATAATAATAACAACAATTTGCCTC
TTCTTTC
CACCTTTTCAGTCTTACTCTCCCTTCTGACATTGAACGCCTCAATCAGTCAGTCGCCTTGTACTTGGCACGGTAATCCT
CCGTGTTC
TTGATATCCTCAGGGGTAGCAAAGCCCTTCATGCCATCGATAATGTCATCCAGAGTGAGGATGGCAAAGATGGGGATGC
CGTACT
CCTTCCTCAGCTCGCCAATGGCACTCGGTCCAGGCTTGGAGTCGTCGCCATCCGCAGCGGGGAGCTTCTCCATGCGGTC
CAGGGCC
ACGACGATGCCGGCGACGATGCCGCCCTCCTTGGTGATCTTCTCAATGGCGTCCCTCTTGGCGGTGCCGGCGGTGATGA
CGTCGTC
GACAATCAGGACCCTCTTGCCCTTGAGCGAAGCGCCGACGATGTTGCCGCCCTCGCCGTGGTCCTTGGCCTCCTTGCGG
TCAAACG
AGTAGGAGACGCGGTCCAGGTTCTGGGGCGCCAGCTCGCCGAGCTTGATGGTGATGGCGGAGCACAGCGGGATGCCCTT
GTAGG
CCGGGCCGAAGACGATGTCGAACTCTAGGCCGGCCTTCTCCTGGGCCTCGATGATGGTCTTTGCAAAGGCGGAGGCGAT
GGCGCC
GGCGAGGCGCGCCGTGTGGAATTCGCCCGCGTTGAAGAAGTAGGGGGATATCCGCTTGGACTTGAGCTCGAAGCTGCCA
AACTTG
AGGACGCCGCCGTCGATGGCGGATTTGAGGAAGTCCTGCTTGTAGGCAGGCAGCTGGGAGGTGGTAGCCATTCTGTTGG
ATTTGG
ATAGTGTCCTTATTCTCTGATTTGAACAGTAGATCAGGACGAGTGAGAGGGATGCAGAGGTTGGATTGGAGTGGTTGAG
CTATAA
AATTTAGAGGCGCGCCGTATCGAGTTTTCACATGGAAGTCAAAGCGTACAGTGCGAGCTTGTACGTTGGTCTTAGTATC
CCACAA
GCTTCTGTCTAGGTATGATGATGGCTATAAGTCACCCAAGGCAGAACTCATCTTGAAGATTGTCTAGAGTGATTTTACC
GCTGATG
AAATGACTGGACTCCCTCCTCCTGCTCTTATACGAAAAATTGCCTGACTCTGCAAAGGTTGTTTGTCTTGGAAGATGAT
GTGCCCC
CCCATCGCTCTTATCTCATACCCCGCCATCTTTCTAGATTCTCATCTTCAACAAGAGGGGCAATCCATGATCTGCGATC
CAGATGT
GCTTCTGGCCTCATACTCTGCCTTCAGGTTGATGTTCACTTAATTGGTGACGAATTCAGCTGATTTGCTGCAGTATGCT
TTGTGTTG
GTTCTTTCCAGGCTTGTGCCAGCCATGAGCGCTTTGAGAGCATGTTGTCACCTATAAACTCGAGTAACGGCCACATATT
GTTCACT
ACTTGAATCACATACCTAATTTTGATAGAATTGACATGTTTAAAGAGCTGAGGTAGCTTTAATGCCTCTGAAGTATTGT
GACACAG
CTTCTCACAGAGTGAGAATGAAAAGTTGGACTCCCCCTAATGAAGTAAAAGTTTCGTCTCTGAACGGTGAAGAGCATAG
ATCCGG
CATCAACTACCTGGCTAGACTACGACGTCAATTCTGCGGCCTTTTGACCTTTATATATGTCCATTAATGCAATAGATTC
TTTTTTTT
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCCCAATTTCGCAGATCAAAGTGGACGTTATAGCATCATAACTAAG
CTCAGTTGCT
GAGGGAAGCCGTCTACTACCTTAGCCCATCCATCCAGCTCCATACCTTGATACTTTAGACGTGAAGCAATTCACACTGT
ACGTCTC
GCAGCTCTCCTTCCCGCTCTTGCTTCCCCACTGGGGTCCATGGTGCGTGTATCGTCCCCTCCTTAATTAAGGCCATTTA
GGCCGTTG
CTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA
CAGGAC
TATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCT
GTCCGCC
TTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCA
AGCTGGGC
TGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGAC
ACGACTT
ATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGG
TGGCCT
AACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTA
GCTCTT
GATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATC
TCAAGA
AGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGCCTGCAGGGCCGATTTTGG
TCATGAG
ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAA
ACTTGGT
CTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACT
CCCCGTCG
TGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGC
TCCAGA
TTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCT
ATTAAT
TGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGG
TGTCACG
CT CGTCGTTTGGTATGGC TTCATTC AGCTC CGGTTCCCAAC GATCAAGGCGAGTTACATGATCCC
CCATGTTGTGCAAAAAAGCGG
TTAGCTC CTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCC
GCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTC
TTACTGTCATGCCATC
CGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGC GACCG
AGTTGCTCTTGCCCGGCGTCAATAC GGGATAATACCGCGC
CACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTC
GGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACC CACTC GTGCAC
CCAACTGATCTTCAGCATCTT
TTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA
ATGTT
GAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGGCCATTTAGGCCT
(Seq ID no 14)
[00298] SEQ ID NO: 15 = Nucleotide sequence of the pTrex8gM TR1037 expression construct.
CTAGAGTTGTGAAGTC GGTAATC CCGCTGTATAGTAATAC GAGTCGCATCTAAATACTCC
GAAGCTGCTGCGAACCCGGAGAATC
GAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAA
TCATGG
CGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTC CCGAAAAAACTC GGAGATTC
CTAAGTAGCGAT
GGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAAC
TGTTCC
GTAC CCCACCTCTTCTCAACCTTTGGC GTTT CC CTGATTCAGC GTACC
CGTACAAGTCGTAATCACTATTAACCCAGACTGACC GG
ACGTGTTTTGC CCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGAC
CGACTGGGGCTGTTC GAAGCCCGA
ATGTAGGATTGTTATC CGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGC
CTCGAAGGTTCACGGCAA
GGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCC GCAATGCAGCATCACTGGAAAATACAAAC
CAATGGCTAAAA
GTACATAAGTTAATGC CTAAAGAAGTCATATAC
CAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAA
CGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGG
TGATCCC
CCAATTGGGTCGCTTGTTTGTTCC GGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTC
GGAGCGTTTTGCATACAACCAA
GGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAG
GTTTGT
CTGCCGATACGACGAATACTGTATAGTCACTTCTGATGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCA
AAAGAT
TGAGTTGAAAC TGCC TAAGAT CT CGGGCCCT CGGGCC TTC GGCCTTTGGGTGTACATGTTTGTGCTCC
GGGCAAATGCAAAGTGTG
GTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGT
TTCGAA
TAGAAAGAGAAGCTTAGCCAAGAACAATAGC CGATAAAGATAGC CTCATTAAAC
GGAATGAGCTAGTAGGCAAAGTCAGCGAAT
GTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGAC
TTGTACA
CCATCTTTTGAGGCACAGAAACCCAATAGTCAAC CATCACAAGTTTGTACAAAAAAGCAGGCTTCACCATGCAGAC
CTTCGGTGC
TTTTCTCGTTTC CTTCCTC GCCGCCAGGTAAGTTGGCCTTGATGAACCATATCATATATCGCC GAGAAGTGGAC
CGCGTGCTGAGA
CT GAGACAGCGGC CTGGCCGC GGCC GAGGGACTTGGAAACCAC GGCCGAAAGC TCGACCCCAACAA GTTC
ACCAAGGATATC AA
GCTCAAGGACCTCCTCAAGGGCAGCCAGAAGCTCGAAGATTTC GCCTACGCCTACC
CCGAGCGCAACCGCGTCTTTGGCGGCAAG
GCCCACCAGGACACCGTCAACTGGATCTACAACGAGCTGAAGAAGACCGGCTACTACGACGTCTACAAGCAGCCCCAGG
TCCAC
CT CTGGT CCAAC GCCGAGCAGAGCCTCAC CGTC GATGGCGAGGCCATCGAC
GCCACCACCATGACCTACAGCCCCAGCCTCAAGG
AAACCAC CGCC GAGGTCGTCGTC GTCCCTGGCCTTGGCTGCACTGCCGCCGACTACC CTGCTGACGTCGCC
GGCAAGATCGCCCTC
ATTCAGCGCGGCAGCTGCACCTTCGGCGAGAAGTCC GTCTACGCCGCTGC CGCCAACGCCGCTGCTGCCATC
GTCTACAACAAC G
TCGAC GGCAGC CTCAGC GGCAC CCTCGGC GCTGC TACTT CT GAGC TGGGCC CCTACGCCCC
CATCGTCGGCATTT CT CTCGC CGAC
GGCCAGAACCTC GTCAGCCTCGCTCAGGCTGGC CCCCTGACCGTCGAC
CTCTACATCAACAGCCAGATGGAAAACCGCACCACCC
ACAACGTCATTGCCAAGAGCAAGGGCGGCGACCCTAACAACGTCATCGTCATCGGCGGCCACAGCGACGCCGTCAACCA
GGGAC
CTGGCGTCAAC GATGAC GGCAGCGGCAT CAT CAGCAACC TCGTGATC
GCCAAGGCCCTCACCAAGTACAGCCTCAAGAACAGCGT
CACCTGGGCCTTTTGGAC CGCCGAAGAGTTCGGCCTCCTC GGCAGCGAGTTCTACGTCAACAGCCTCTCTGCC
GCCGAGAAGGAC
AAGATCAAGCTCTACCTCAACTTC GACATGATCGC CAGCCCCAACTACGCCCTCATGATCTACGAC
GGCGACGGCAGCACCTTCA
ACATGACCGGC CCTGC CGGCTC CGCCGAGATCGAGCACCTCTTCGAGGACTACTACAAGTCTC
GCGGCCTCAGCTACATC CCCAC
CGCCTTTGACGGCC GCAGCGACTACGAGGC CTTCATCCTCAAC GGCATCCCC GCTGGCGGC
CTCTTCACTGGCGC CGAGCAGATC
AAGACCGAGGAACAGGTCGCCATGTTCGGCGGCCAGGCTGGCGTCGCCTACGACCCCAACTATCACGCCGCTGGCGACA
ACATG
ACCAACCTCAGCGAGGAAGCCTTCCTCATCAACAGCAAGGCCACCGCCTTCGCCGTCGCCACCTACGCCAACAGCCTTG
AGAGCA
TCCCCCCTCGCAACGCCACCATGAGCATCCAGACCCGCTCTGCCTCTCGCCGAGCCGCTGCTCATCGACGAGCCGCCAA
GCCTCAC
TCTCACTCTGGCGGCACTGGCTGCTGGCACACCCGAGTCGAGCTGTAAGACCCAGCTTTCTTGTACAAAGTGGTGATCG
CGCCAG
CTCCGTGCGAAAGCCTGACGCACCGGTAGATTCTTGGTGAGCCCGTATCATGACGGCGGCGGGAGCTACATGGCCCCGG
GTGATT
TATTTTTTTTGTATCTACTTCTGACCCTTTTCAAATATACGGTCAACTCATCTTTCACTGGAGATGCGGCCTGCTTGGT
ATTGCGATG
TTGTCAGCTTGGCAAATTGTGGCTTTCGAAAACACAAAACGATTCCTTAGTAGCCATGCATTTTAAGATAACGGAATAG
AAGAAA
GAGGAAATTAAAAAAAAAAAAAAAACAAACATCCCGTTCATAACCCGTAGAATCGCCGCTCTTCGGCTAGCTAGTTACG
CTTGTT
TATTTACGACAAGATCTAGAAGATTCGAGATAGAATAATAATAATAACAACAATTTGCCTCTTCTTTCCACCTTTTCAG
TCTTACTC
TCCCTTCTGACATTGAACGCCTCAATCAGTCAGTCGCCTTGTACTTGGCACGGTAATCCTCCGTGTTCTTGATATCCTC
AGGGGTAG
CAAAGCCCTTCATGCCATCGATAATGTCATCCAGAGTGAGGATGGCAAAGATGGGGATGCCGTACTCCTTCCTCAGCTC
GCCAAT
GGCACTCGGTCCAGGCTTGGAGTCGTCGCCATCCGCAGCGGGGAGCTTCTCCATGCGGTCCAGGGCCACGACGATGCCG
GCGACG
ATGCCGCCCTCCTTGGTGATCTTCTCAATGGCGTCCCTCTTGGCGGTGCCGGCGGTGATGACGTCGTCGACAATCAGGA
CCCTCTT
GCCCTTGAGCGAAGCGCCGACGATGTTGCCGCCCTCGCCGTGGTCCTTGGCCTCCTTGCGGTCAAACGAGTAGGAGACG
CGGTCC
AGGTTCTGGGGCGCCAGCTCGCCGAGCTTGATGGTGATGGCGGAGCACAGCGGGATGCCCTTGTAGGCCGGGCCGAAGA
CGATG
TCGAACTCTAGGCCGGCCTTCTCCTGGGCCTCGATGATGGTCTTTGCAAAGGCGGAGGCGATGGCGCCGGCGAGGCGCG
CCGTGT
GGAATTCGCCCGCGTTGAAGAAGTAGGGGGATATCCGCTTGGACTTGAGCTCGAAGCTGCCAAACTTGAGGACGCCGCC
GTCGAT
GGCGGATTTGAGGAAGTCCTGCTTGTAGGCAGGCAGCTGGGAGGTGGTAGCCATTCTGTTGGATTTGGATAGTGTCCTT
ATTCTCT
GATTTGAACAGTAGATCAGGACGAGTGAGAGGGATGCAGAGGTTGGATTGGAGTGGTTGAGCTATAAAATTTAGAGGCG
CGCCG
TATCGAGTTTTCACATGGAAGTCAAAGCGTACAGTGCGAGCTTGTACGTTGGTCTTAGTATCCCACAAGCTTCTGTCTA
GGTATGA
TGATGGCTATAAGTCACCCAAGGCAGAACTCATCTTGAAGATTGTCTAGAGTGATTTTACCGCTGATGAAATGACTGGA
CTCCCTC
CTCCTGCTCTTATACGAAAAATTGCCTGACTCTGCAAAGGTTGTTTGTCTTGGAAGATGATGTGCCCCCCCATCGCTCT
TATCTCAT
ACCCCGCCATCTTTCTAGATTCTCATCTTCAACAAGAGGGGCAATCCATGATCTGCGATCCAGATGTGCTTCTGGCCTC
ATACTCT
GCCTTCAGGTTGATGTTCACTTAATTGGTGACGAATTCAGCTGATTTGCTGCAGTATGCTTTGTGTTGGTTCTTTCCAG
GCTTGTGC
CAGCCATGAGCGCTTTGAGAGCATGTTGTCACCTATAAACTCGAGTAACGGCCACATATTGTTCACTACTTGAATCACA
TACCTAA
TTTTGATAGAATTGACATGTTTAAAGAGCTGAGGTAGCTTTAATGCCTCTGAAGTATTGTGACACAGCTTCTCACAGAG
TGAGAAT
GAAAAGTTGGACTCCCCCTAATGAAGTAAAAGTTTCGTCTCTGAACGGTGAAGAGCATAGATCCGGCATCAACTACCTG
GCTAGA
CTACGACGTCAATTCTGCGGCCTTTTGACCTTTATATATGTCCATTAATGCAATAGATTCTTTTTTTTTTTTTTTTTTT
TTTTTTTTTTT
TTTTTTTTTTTGCCCAATTTCGCAGATCAAAGTGGACGTTATAGCATCATAACTAAGCTCAGTTGCTGAGGGAAGCCGT
CTACTAC
CTTAGCCCATCCATCCAGCTCCATACCTTGATACTTTAGACGTGAAGCAATTCACACTGTACGTCTCGCAGCTCTCCTT
CCCGCTCT
TGCTTCCCCACTGGGGTCCATGGTGCGTGTATCGTCCCCTCCTTAATTAAGGCCATTTAGGCCGTTGCTGGCGTTTTTC
CATAGGCT
CCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAG
GCGTT
TCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCG
GGAAGCGT
GGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAA
CCCCCCG
TTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGC
AGCAGCC
ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACA
CTAGAA
GGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACA
AACCAC
CGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATC
TTTTCTA
CGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGCCTGCAGGGCCGATTTTGGTCATGAGATTATCAAAAAGG
ATCTTC
ACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACC
AATGCTT
AATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACT
ACGATAC
GGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAAT
AAACCA
GCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAA
GCTAGA
GTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTG
GTATGGC
TTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTC
GGTCCTC
CGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCAT
GCCATCC
GTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTT
GCCCGGC
GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAA
CTCTCA
AGGATCTTAC CGCTGTTGAGATCCAGTTC GATGTAACCCACTCGTGCACC
CAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTT
TCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATAC
TCTTC
CTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGGCCATTTAGGCCT (Seq ID no 15)
CTAGAGTTGTGAAGTC GGTAATC CCGCTGTATAGTAATAC GAGTCGCATCTAAATACTCC
GAAGCTGCTGCGAACCCGGAGAATC
GAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAA
TCATGG
CGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTC CCGAAAAAACTC GGAGATTC
CTAAGTAGCGAT
GGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAAC
TGTTCC
GTAC CCCACCTCTTCTCAACCTTTGGC GTTT CC CTGATTCAGC GTACC
CGTACAAGTCGTAATCACTATTAACCCAGACTGACC GG
ACGTGTTTTGC CCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGAC
CGACTGGGGCTGTTC GAAGCCCGA
ATGTAGGATTGTTATC CGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGC
CTCGAAGGTTCACGGCAA
GGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCC GCAATGCAGCATCACTGGAAAATACAAAC
CAATGGCTAAAA
GTACATAAGTTAATGC CTAAAGAAGTCATATAC
CAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAA
CGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGG
TGATCCC
CCAATTGGGTCGCTTGTTTGTTCC GGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTC
GGAGCGTTTTGCATACAACCAA
GGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAG
GTTTGT
CTGCCGATACGACGAATACTGTATAGTCACTTCTGATGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCA
AAAGAT
TGAGTTGAAAC TGCC TAAGAT CT CGGGCCCT CGGGCC TTC GGCCTTTGGGTGTACATGTTTGTGCTCC
GGGCAAATGCAAAGTGTG
GTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGT
TTCGAA
TAGAAAGAGAAGCTTAGCCAAGAACAATAGC CGATAAAGATAGC CTCATTAAAC
GGAATGAGCTAGTAGGCAAAGTCAGCGAAT
GTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGAC
TTGTACA
CCATCTTTTGAGGCACAGAAACCCAATAGTCAAC CATCACAAGTTTGTACAAAAAAGCAGGCTTCACCATGCAGAC
CTTCGGTGC
TTTTCTCGTTTC CTTCCTC GCCGCCAGGTAAGTTGGCCTTGATGAACCATATCATATATCGCC GAGAAGTGGAC
CGCGTGCTGAGA
CT GAGACAGCGGC CTGGCCGC GGCC GAGGGACTTGGAAACCAC GGCCGAAAGC TCGACCCCAACAA GTTC
ACCAAGGATATC AA
GCTCAAGGACCTCCTCAAGGGCAGCCAGAAGCTCGAAGATTTC GCCTACGCCTACC
CCGAGCGCAACCGCGTCTTTGGCGGCAAG
GCCCACCAGGACACCGTCAACTGGATCTACAACGAGCTGAAGAAGACCGGCTACTACGACGTCTACAAGCAGCCCCAGG
TCCAC
CT CTGGT CCAAC GCCGAGCAGAGCCTCAC CGTC GATGGCGAGGCCATCGAC
GCCACCACCATGACCTACAGCCCCAGCCTCAAGG
AAACCAC CGCC GAGGTCGTCGTC GTCCCTGGCCTTGGCTGCACTGCCGCCGACTACC CTGCTGACGTCGCC
GGCAAGATCGCCCTC
ATTCAGCGCGGCAGCTGCACCTTCGGCGAGAAGTCC GTCTACGCCGCTGC CGCCAACGCCGCTGCTGCCATC
GTCTACAACAAC G
TCGAC GGCAGC CTCAGC GGCAC CCTCGGC GCTGC TACTT CT GAGC TGGGCC CCTACGCCCC
CATCGTCGGCATTT CT CTCGC CGAC
GGCCAGAACCTC GTCAGCCTCGCTCAGGCTGGC CCCCTGACCGTCGAC
CTCTACATCAACAGCCAGATGGAAAACCGCACCACCC
ACAACGTCATTGCCAAGAGCAAGGGCGGCGACCCTAACAACGTCATCGTCATCGGCGGCCACAGCGACGCCGTCAACCA
GGGAC
CTGGCGTCAAC GATGAC GGCAGCGGCAT CAT CAGCAACC TCGTGATC
GCCAAGGCCCTCACCAAGTACAGCCTCAAGAACAGCGT
CACCTGGGCCTTTTGGAC CGCCGAAGAGTTCGGCCTCCTC GGCAGCGAGTTCTACGTCAACAGCCTCTCTGCC
GCCGAGAAGGAC
AAGATCAAGCTCTACCTCAACTTC GACATGATCGC CAGCCCCAACTACGCCCTCATGATCTACGAC
GGCGACGGCAGCACCTTCA
ACATGACCGGC CCTGC CGGCTC CGCCGAGATCGAGCACCTCTTCGAGGACTACTACAAGTCTC
GCGGCCTCAGCTACATC CCCAC
CGCCTTTGACGGCC GCAGCGACTACGAGGC CTTCATCCTCAAC GGCATCCCC GCTGGCGGC
CTCTTCACTGGCGC CGAGCAGATC
AAGACCGAGGAACAGGTCGCCATGTTCGGCGGCCAGGCTGGCGTCGCCTACGACCCCAACTATCACGCCGCTGGCGACA
ACATG
ACCAACCTCAGCGAGGAAGCCTTCCTCATCAACAGCAAGGCCACCGCCTTCGCCGTCGCCACCTACGCCAACAGCCTTG
AGAGCA
TCCCCCCTCGCAACGCCACCATGAGCATCCAGACCCGCTCTGCCTCTCGCCGAGCCGCTGCTCATCGACGAGCCGCCAA
GCCTCAC
TCTCACTCTGGCGGCACTGGCTGCTGGCACACCCGAGTCGAGCTGTAAGACCCAGCTTTCTTGTACAAAGTGGTGATCG
CGCCAG
CTCCGTGCGAAAGCCTGACGCACCGGTAGATTCTTGGTGAGCCCGTATCATGACGGCGGCGGGAGCTACATGGCCCCGG
GTGATT
TATTTTTTTTGTATCTACTTCTGACCCTTTTCAAATATACGGTCAACTCATCTTTCACTGGAGATGCGGCCTGCTTGGT
ATTGCGATG
TTGTCAGCTTGGCAAATTGTGGCTTTCGAAAACACAAAACGATTCCTTAGTAGCCATGCATTTTAAGATAACGGAATAG
AAGAAA
GAGGAAATTAAAAAAAAAAAAAAAACAAACATCCCGTTCATAACCCGTAGAATCGCCGCTCTTCGGCTAGCTAGTTACG
CTTGTT
TATTTACGACAAGATCTAGAAGATTCGAGATAGAATAATAATAATAACAACAATTTGCCTCTTCTTTCCACCTTTTCAG
TCTTACTC
TCCCTTCTGACATTGAACGCCTCAATCAGTCAGTCGCCTTGTACTTGGCACGGTAATCCTCCGTGTTCTTGATATCCTC
AGGGGTAG
CAAAGCCCTTCATGCCATCGATAATGTCATCCAGAGTGAGGATGGCAAAGATGGGGATGCCGTACTCCTTCCTCAGCTC
GCCAAT
GGCACTCGGTCCAGGCTTGGAGTCGTCGCCATCCGCAGCGGGGAGCTTCTCCATGCGGTCCAGGGCCACGACGATGCCG
GCGACG
ATGCCGCCCTCCTTGGTGATCTTCTCAATGGCGTCCCTCTTGGCGGTGCCGGCGGTGATGACGTCGTCGACAATCAGGA
CCCTCTT
GCCCTTGAGCGAAGCGCCGACGATGTTGCCGCCCTCGCCGTGGTCCTTGGCCTCCTTGCGGTCAAACGAGTAGGAGACG
CGGTCC
AGGTTCTGGGGCGCCAGCTCGCCGAGCTTGATGGTGATGGCGGAGCACAGCGGGATGCCCTTGTAGGCCGGGCCGAAGA
CGATG
TCGAACTCTAGGCCGGCCTTCTCCTGGGCCTCGATGATGGTCTTTGCAAAGGCGGAGGCGATGGCGCCGGCGAGGCGCG
CCGTGT
GGAATTCGCCCGCGTTGAAGAAGTAGGGGGATATCCGCTTGGACTTGAGCTCGAAGCTGCCAAACTTGAGGACGCCGCC
GTCGAT
GGCGGATTTGAGGAAGTCCTGCTTGTAGGCAGGCAGCTGGGAGGTGGTAGCCATTCTGTTGGATTTGGATAGTGTCCTT
ATTCTCT
GATTTGAACAGTAGATCAGGACGAGTGAGAGGGATGCAGAGGTTGGATTGGAGTGGTTGAGCTATAAAATTTAGAGGCG
CGCCG
TATCGAGTTTTCACATGGAAGTCAAAGCGTACAGTGCGAGCTTGTACGTTGGTCTTAGTATCCCACAAGCTTCTGTCTA
GGTATGA
TGATGGCTATAAGTCACCCAAGGCAGAACTCATCTTGAAGATTGTCTAGAGTGATTTTACCGCTGATGAAATGACTGGA
CTCCCTC
CTCCTGCTCTTATACGAAAAATTGCCTGACTCTGCAAAGGTTGTTTGTCTTGGAAGATGATGTGCCCCCCCATCGCTCT
TATCTCAT
ACCCCGCCATCTTTCTAGATTCTCATCTTCAACAAGAGGGGCAATCCATGATCTGCGATCCAGATGTGCTTCTGGCCTC
ATACTCT
GCCTTCAGGTTGATGTTCACTTAATTGGTGACGAATTCAGCTGATTTGCTGCAGTATGCTTTGTGTTGGTTCTTTCCAG
GCTTGTGC
CAGCCATGAGCGCTTTGAGAGCATGTTGTCACCTATAAACTCGAGTAACGGCCACATATTGTTCACTACTTGAATCACA
TACCTAA
TTTTGATAGAATTGACATGTTTAAAGAGCTGAGGTAGCTTTAATGCCTCTGAAGTATTGTGACACAGCTTCTCACAGAG
TGAGAAT
GAAAAGTTGGACTCCCCCTAATGAAGTAAAAGTTTCGTCTCTGAACGGTGAAGAGCATAGATCCGGCATCAACTACCTG
GCTAGA
CTACGACGTCAATTCTGCGGCCTTTTGACCTTTATATATGTCCATTAATGCAATAGATTCTTTTTTTTTTTTTTTTTTT
TTTTTTTTTTT
TTTTTTTTTTTGCCCAATTTCGCAGATCAAAGTGGACGTTATAGCATCATAACTAAGCTCAGTTGCTGAGGGAAGCCGT
CTACTAC
CTTAGCCCATCCATCCAGCTCCATACCTTGATACTTTAGACGTGAAGCAATTCACACTGTACGTCTCGCAGCTCTCCTT
CCCGCTCT
TGCTTCCCCACTGGGGTCCATGGTGCGTGTATCGTCCCCTCCTTAATTAAGGCCATTTAGGCCGTTGCTGGCGTTTTTC
CATAGGCT
CCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAG
GCGTT
TCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCG
GGAAGCGT
GGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAA
CCCCCCG
TTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGC
AGCAGCC
ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACA
CTAGAA
GGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACA
AACCAC
CGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATC
TTTTCTA
CGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGCCTGCAGGGCCGATTTTGGTCATGAGATTATCAAAAAGG
ATCTTC
ACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACC
AATGCTT
AATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACT
ACGATAC
GGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAAT
AAACCA
GCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAA
GCTAGA
GTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTG
GTATGGC
TTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTC
GGTCCTC
CGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCAT
GCCATCC
GTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTT
GCCCGGC
GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAA
CTCTCA
AGGATCTTAC CGCTGTTGAGATCCAGTTC GATGTAACCCACTCGTGCACC
CAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTT
TCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATAC
TCTTC
CTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGGCCATTTAGGCCT (Seq ID no 15)
[00299] SEQ ID NO: 16 = Nucleotide sequence of the pTrex8gM TRI038 expression construct.
CTAGAGTTGTGAAGTC GGTAATC CCGCTGTATAGTAATAC GAGTCGCATCTAAATACTCC
GAAGCTGCTGCGAACCCGGAGAATC
GAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAA
TCATGG
CGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTC CCGAAAAAACTC GGAGATTC
CTAAGTAGCGAT
GGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAAC
TGTTCC
GTAC CCCACCTCTTCTCAACCTTTGGC GTTTCC CTGATTCAGC GTACC
CGTACAAGTCGTAATCACTATTAACCCAGACTGACC GG
ACGTGTTTTGC CCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGAC
CGACTGGGGCTGTTC GAAGCCCGA
ATGTAGGATTGTTATC CGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGC
CTCGAAGGTTCACGGCAA
GGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCC GCAATGCAGCATCACTGGAAAATACAAAC
CAATGGCTAAAA
GTACATAAGTTAATGC CTAAAGAAGTCATATAC
CAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAA
CGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGG
TGATCCC
CCAATTGGGTCGCTTGTTTGTTCC GGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTC
GGAGCGTTTTGCATACAACCAA
GGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAG
GTTTGT
CTGCCGATACGACGAATACTGTATAGTCACTTCTGATGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCA
AAAGAT
TGAGTTGAAACTGCCTAAGATCTCGGGCCCTCGGGCCTTC GGCCTTTGGGTGTACATGTTTGTGCTCC
GGGCAAATGCAAAGTGTG
GTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGT
TTCGAA
TAGAAAGAGAAGCTTAGCCAAGAACAATAGC CGATAAAGATAGC CTCATTAAAC
GGAATGAGCTAGTAGGCAAAGTCAGCGAAT
GTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGAC
TTGTACA
CCATCTTTTGAGGCACAGAAACCCAATAGTCAAC CATCACAAGTTTGTACAAAAAAGCAGGCTTCACCATGCAGAC
CTTCGGTGC
TTTTCTCGTTTC CTTCCTC GCCGCCAGGTAAGTTGGCCTTGATGAACCATATCATATATCGCC GAGAAGTGGAC
CGCGTGCTGAGA
CT GAGACAGCGGC CTGGCC GCGGC CGGCAAGCACAAGCCTCTTGTCAC
CCCTGAGGCCCTCCAGGACCTGATTACCCTCGACGAC
CTCCTCGC CGGCAGC CAGCAGCTCCAGGACTTC GCCTACGCCTACC CCGAGCGCAACCGC GTCTTTGGC
GGCCGAGCCCAC GACG
ACACC GTCAACTGGCTCTACCGCGAGCTGAAGC GCACCGGCTACTACCAC GTCTACAAGCAGC
CCCAGGTCCACCTCTACAGC AA
CGCC GAGGAAAGCCTCACC GTCAAC GGCGAGGC CATCGAGGCCACCACCATGAC CTACAGC
CCCAGCGCCAACGC CTCTGCCGA
GCTGGCTGTCATCAGCGGCCTTGGCTGCTCTCC CGCCGACTTC GCCTCTGACGTC GCCGGCAAGGTCGTCCTC
GTCCAGCGAGGCA
ACT GCACCTTCGGCGAGAAGTCCGTCTACGCC GCTGCCGCCGATGCCGC CGCTACGATC GTCTACAACAAC
GTCGAGGGCAGCCT
CAGCGGCACCCTCGGCGCTGCTCAGTCTGAGCAAGGCCCCTACAGCGGCATC GTC GGCATCAGCCTCGCTGAC
GGCGAGGC CCTC
CTCGCCCTTGCTGAGGAAGGC CCTGTCCACGTCGAC
CTCTGGATCGACAGCGTCATGGAAAACCGCACCACCTACAACGTCATTG
CCCAGACCAAGGGC GGCGACC CCGACAAC GTCGTCACTCTTGGCGGC CACAGCGACAGCGTC
GAGGCTGGCCCTGGCATCAACG
ACGACGGCAGCGGCATCATCAGCAAC CTCGTCATTGCCC GAGCC CTCACCAAGTTCAGCACCAAGCACGC
CGTCCGCTTTTTCTTC
TGGAC CGCCGAAGAGTTCGGCCTCCTC GGCAGC GACTACTACGTCAGCAGCCTCAGCCC
CGCTGAGCTGGCCAAGATCCGCCTCT
ACCTCAACTTCGACATGATCGCCAGCCCCAACTACGGC CTCCTCCTCTACGATGGC GACGGCAGC GCCTTCAAC
CTCACTGGCC CT
GCTGGCAGCGAC GCCATCGAGAAGCTGTTCTACGACTACTTC
CAGAGCATCGGCCAGGCCACCGTCGAGACTGAGTTCGAC GGCC
GCAGCGACTAC GAGGCCTTCATCCTCAACGGCATCCC CGCTGGC GGCGTCTTTACTGGCGCC
GAGGAAATCAAGAGC GAGGAAG
AGGTC GCCCTCTGGGGCGGAGAGGCTGGCGTCGCCTACGACGC CAACTACCAC CAGGTC GGCGACACCATC
GACAAC CTCAACA
CCGAGGCCTAC CTGCTCAACAGCAAGGCCACCGCCTTCGC CGTC GCCAC CTACGC CAACGAC
CTCAGCACCATCCC CAAGCGC GA
GATGACCACCGCCGTCAAGCGAGCCAACGTCAAC GGCCACATGCACC GCCGCAC CATGC
CCAAGAAGCGCCAGACTGC CCACCG
CCACGCTGCCAAGGGCTGCTTTCACAGCCGCGTCGAGCAGTAAGACCCAGCTTTCTTGTACAAAGTGGTGATCGCGCCA
GCTCCG
TGC GAAAGCCTGACGCAC CGGTAGATTCTTGGTGAGCC CGTATCATGAC GGCGGC GGGAGCTACATGGCCC
CGGGTGATTTATTT
TTTTTGTATCTACTTCTGACCCTTTTCAAATATACGGTCAACTCATCTTTCACTGGAGATGCGGCCTGCTTGGTATTGC
GATGTTGT
CAGCTTGGCAAATTGTGGCTTTCGAAAACACAAAACGATTCCTTAGTAGCCATGCATTTTAAGATAACGGAATAGAAGA
AAGAGG
AAATTAAAAAAAAAAAAAAAACAAACATCCCGTTCATAACCCGTAGAATCGCCGCTCTTCGGCTAGCTAGTTACGCTTG
TTTATTT
ACGACAAGATCTAGAAGATTCGAGATAGAATAATAATAATAACAACAATTTGCCTCTTCTTTCCACCTTTTCAGTCTTA
CTCTCCC
TTCTGACATTGAACGCCTCAATCAGTCAGTCGCCTTGTACTTGGCACGGTAATCCTCCGTGTTCTTGATATCCTCAGGG
GTAGCAA
AGCCCTTCATGCCATCGATAATGTCATCCAGAGTGAGGATGGCAAAGATGGGGATGCCGTACTCCTTCCTCAGCTCGCC
AATGGC
ACTCGGTCCAGGCTTGGAGTC GTCGC CATCCGCAGCGGGGAGCTTCTC CATGC GGTCCAGGGCCAC
GACGATGC CGGCGAC GATG
CCGC CCTC CTTGGTGATCTTCTCAATGGCGTCC CTCTTGGC
GGTGCCGGCGGTGATGACGTCGTCGACAATCAGGAC CCTCTTGCC
CTTGAGCGAAGCGCCGACGATGTTGCCGCCCTCGCCGTGGTCCTTGGCCTCCTTGCGGTCAAACGAGTAGGAGACGCGG
TCCAGG
TTCTGGGGCGCCAGCTCGCCGAGCTTGATGGTGATGGCGGAGCACAGCGGGATGCCCTTGTAGGCCGGGCCGAAGACGA
TGTCG
AACTCTAGGCCGGCCTTCTCCTGGGCCTCGATGATGGTCTTTGCAAAGGCGGAGGCGATGGCGCCGGCGAGGCGCGCCG
TGTGGA
ATTCGCCCGCGTTGAAGAAGTAGGGGGATATCCGCTTGGACTTGAGCTCGAAGCTGCCAAACTTGAGGACGCCGCCGTC
GATGGC
GGATTTGAGGAAGTCCTGCTTGTAGGCAGGCAGCTGGGAGGTGGTAGCCATTCTGTTGGATTTGGATAGTGTCCTTATT
CTCTGAT
TTGAACAGTAGATCAGGACGAGTGAGAGGGATGCAGAGGTTGGATTGGAGTGGTTGAGCTATAAAATTTAGAGGCGCGC
CGTAT
CGAGTTTTCACATGGAAGTCAAAGCGTACAGTGCGAGCTTGTACGTTGGTCTTAGTATCCCACAAGCTTCTGTCTAGGT
ATGATGA
TGGCTATAAGTCACCCAAGGCAGAACTCATCTTGAAGATTGTCTAGAGTGATTTTACCGCTGATGAAATGACTGGACTC
CCTCCTC
CTGCTCTTATACGAAAAATTGCCTGACTCTGCAAAGGTTGTTTGTCTTGGAAGATGATGTGCCCCCCCATCGCTCTTAT
CTCATACC
CCGCCATCTTTCTAGATTCTCATCTTCAACAAGAGGGGCAATCCATGATCTGCGATCCAGATGTGCTTCTGGCCTCATA
CTCTGCCT
TCAGGTTGATGTTCACTTAATTGGTGACGAATTCAGCTGATTTGCTGCAGTATGCTTTGTGTTGGTTCTTTCCAGGCTT
GTGCCAGC
CATGAGCGCTTTGAGAGCATGTTGTCACCTATAAACTCGAGTAACGGCCACATATTGTTCACTACTTGAATCACATACC
TAATTTT
GATAGAATTGACATGTTTAAAGAGCTGAGGTAGCTTTAATGCCTCTGAAGTATTGTGACACAGCTTCTCACAGAGTGAG
AATGAA
AAGTTGGACTCCCCCTAATGAAGTAAAAGTTTCGTCTCTGAACGGTGAAGAGCATAGATCCGGCATCAACTACCTGGCT
AGACTA
CGACGTCAATTCTGCGGCCTTTTGACCTTTATATATGTCCATTAATGCAATAGATTCTTTTTTTTTTTTTTTTTTTTTT
TTTTTTTTTTT
TTTTTTTTGCCCAATTTCGCAGATCAAAGTGGACGTTATAGCATCATAACTAAGCTCAGTTGCTGAGGGAAGCCGTCTA
CTACCTT
AGCCCATCCATCCAGCTCCATACCTTGATACTTTAGACGTGAAGCAATTCACACTGTACGTCTCGCAGCTCTCCTTCCC
GCTCTTGC
TTCCCCACTGGGGTCCATGGTGCGTGTATCGTCCCCTCCTTAATTAAGGCCATTTAGGCCGTTGCTGGCGTTTTTCCAT
AGGCTCCG
CCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCG
TTTCC
CCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGA
AGCGTGGC
GCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCC
CCCGTTC
AGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGC
AGCCAC
TGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACT
AGAAGG
ACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAA
CCACCG
CTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT
TTCTACG
GGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGCCTGCAGGGCCGATTTTGGTCATGAGATTATCAAAAAGGAT
CTTCAC
CTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAA
TGCTTAA
TCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTAC
GATACGG
GAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAA
ACCAGC
CAGCC GGAAGGGCCGAGC GCAGAAGTGGTCCTGCAACTTTATC CGCCTC CATC CAGTCTATTAATTGTTGCC
GGGAAGCTAGAGT
AAGTAGTTC GCCAGTTAATAGTTTGCGCAAC GTTGTTGC CATTGCTACAGGCATC GTGGTGTCAC GCTC
GTCGTTTGGTATGGC TT
CATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGG
TCCTCCG
ATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGC
CATCCGT
AAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGC
CCGGCGT
CAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACT
CTCAAG
GATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACC
AGCGTTTC
TGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTC
TTCCT
TTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGGCCATTTAGGCCT (Seq ID no 16) Example 2 - Operational p11- and temperature optimum of the PepN 2 from Neosartorya fischeri (TRI031) in gluten hydrolyses
CTAGAGTTGTGAAGTC GGTAATC CCGCTGTATAGTAATAC GAGTCGCATCTAAATACTCC
GAAGCTGCTGCGAACCCGGAGAATC
GAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAA
TCATGG
CGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTC CCGAAAAAACTC GGAGATTC
CTAAGTAGCGAT
GGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAAC
TGTTCC
GTAC CCCACCTCTTCTCAACCTTTGGC GTTTCC CTGATTCAGC GTACC
CGTACAAGTCGTAATCACTATTAACCCAGACTGACC GG
ACGTGTTTTGC CCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGAC
CGACTGGGGCTGTTC GAAGCCCGA
ATGTAGGATTGTTATC CGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGC
CTCGAAGGTTCACGGCAA
GGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCC GCAATGCAGCATCACTGGAAAATACAAAC
CAATGGCTAAAA
GTACATAAGTTAATGC CTAAAGAAGTCATATAC
CAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAA
CGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGG
TGATCCC
CCAATTGGGTCGCTTGTTTGTTCC GGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTC
GGAGCGTTTTGCATACAACCAA
GGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAG
GTTTGT
CTGCCGATACGACGAATACTGTATAGTCACTTCTGATGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCA
AAAGAT
TGAGTTGAAACTGCCTAAGATCTCGGGCCCTCGGGCCTTC GGCCTTTGGGTGTACATGTTTGTGCTCC
GGGCAAATGCAAAGTGTG
GTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGT
TTCGAA
TAGAAAGAGAAGCTTAGCCAAGAACAATAGC CGATAAAGATAGC CTCATTAAAC
GGAATGAGCTAGTAGGCAAAGTCAGCGAAT
GTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGAC
TTGTACA
CCATCTTTTGAGGCACAGAAACCCAATAGTCAAC CATCACAAGTTTGTACAAAAAAGCAGGCTTCACCATGCAGAC
CTTCGGTGC
TTTTCTCGTTTC CTTCCTC GCCGCCAGGTAAGTTGGCCTTGATGAACCATATCATATATCGCC GAGAAGTGGAC
CGCGTGCTGAGA
CT GAGACAGCGGC CTGGCC GCGGC CGGCAAGCACAAGCCTCTTGTCAC
CCCTGAGGCCCTCCAGGACCTGATTACCCTCGACGAC
CTCCTCGC CGGCAGC CAGCAGCTCCAGGACTTC GCCTACGCCTACC CCGAGCGCAACCGC GTCTTTGGC
GGCCGAGCCCAC GACG
ACACC GTCAACTGGCTCTACCGCGAGCTGAAGC GCACCGGCTACTACCAC GTCTACAAGCAGC
CCCAGGTCCACCTCTACAGC AA
CGCC GAGGAAAGCCTCACC GTCAAC GGCGAGGC CATCGAGGCCACCACCATGAC CTACAGC
CCCAGCGCCAACGC CTCTGCCGA
GCTGGCTGTCATCAGCGGCCTTGGCTGCTCTCC CGCCGACTTC GCCTCTGACGTC GCCGGCAAGGTCGTCCTC
GTCCAGCGAGGCA
ACT GCACCTTCGGCGAGAAGTCCGTCTACGCC GCTGCCGCCGATGCCGC CGCTACGATC GTCTACAACAAC
GTCGAGGGCAGCCT
CAGCGGCACCCTCGGCGCTGCTCAGTCTGAGCAAGGCCCCTACAGCGGCATC GTC GGCATCAGCCTCGCTGAC
GGCGAGGC CCTC
CTCGCCCTTGCTGAGGAAGGC CCTGTCCACGTCGAC
CTCTGGATCGACAGCGTCATGGAAAACCGCACCACCTACAACGTCATTG
CCCAGACCAAGGGC GGCGACC CCGACAAC GTCGTCACTCTTGGCGGC CACAGCGACAGCGTC
GAGGCTGGCCCTGGCATCAACG
ACGACGGCAGCGGCATCATCAGCAAC CTCGTCATTGCCC GAGCC CTCACCAAGTTCAGCACCAAGCACGC
CGTCCGCTTTTTCTTC
TGGAC CGCCGAAGAGTTCGGCCTCCTC GGCAGC GACTACTACGTCAGCAGCCTCAGCCC
CGCTGAGCTGGCCAAGATCCGCCTCT
ACCTCAACTTCGACATGATCGCCAGCCCCAACTACGGC CTCCTCCTCTACGATGGC GACGGCAGC GCCTTCAAC
CTCACTGGCC CT
GCTGGCAGCGAC GCCATCGAGAAGCTGTTCTACGACTACTTC
CAGAGCATCGGCCAGGCCACCGTCGAGACTGAGTTCGAC GGCC
GCAGCGACTAC GAGGCCTTCATCCTCAACGGCATCCC CGCTGGC GGCGTCTTTACTGGCGCC
GAGGAAATCAAGAGC GAGGAAG
AGGTC GCCCTCTGGGGCGGAGAGGCTGGCGTCGCCTACGACGC CAACTACCAC CAGGTC GGCGACACCATC
GACAAC CTCAACA
CCGAGGCCTAC CTGCTCAACAGCAAGGCCACCGCCTTCGC CGTC GCCAC CTACGC CAACGAC
CTCAGCACCATCCC CAAGCGC GA
GATGACCACCGCCGTCAAGCGAGCCAACGTCAAC GGCCACATGCACC GCCGCAC CATGC
CCAAGAAGCGCCAGACTGC CCACCG
CCACGCTGCCAAGGGCTGCTTTCACAGCCGCGTCGAGCAGTAAGACCCAGCTTTCTTGTACAAAGTGGTGATCGCGCCA
GCTCCG
TGC GAAAGCCTGACGCAC CGGTAGATTCTTGGTGAGCC CGTATCATGAC GGCGGC GGGAGCTACATGGCCC
CGGGTGATTTATTT
TTTTTGTATCTACTTCTGACCCTTTTCAAATATACGGTCAACTCATCTTTCACTGGAGATGCGGCCTGCTTGGTATTGC
GATGTTGT
CAGCTTGGCAAATTGTGGCTTTCGAAAACACAAAACGATTCCTTAGTAGCCATGCATTTTAAGATAACGGAATAGAAGA
AAGAGG
AAATTAAAAAAAAAAAAAAAACAAACATCCCGTTCATAACCCGTAGAATCGCCGCTCTTCGGCTAGCTAGTTACGCTTG
TTTATTT
ACGACAAGATCTAGAAGATTCGAGATAGAATAATAATAATAACAACAATTTGCCTCTTCTTTCCACCTTTTCAGTCTTA
CTCTCCC
TTCTGACATTGAACGCCTCAATCAGTCAGTCGCCTTGTACTTGGCACGGTAATCCTCCGTGTTCTTGATATCCTCAGGG
GTAGCAA
AGCCCTTCATGCCATCGATAATGTCATCCAGAGTGAGGATGGCAAAGATGGGGATGCCGTACTCCTTCCTCAGCTCGCC
AATGGC
ACTCGGTCCAGGCTTGGAGTC GTCGC CATCCGCAGCGGGGAGCTTCTC CATGC GGTCCAGGGCCAC
GACGATGC CGGCGAC GATG
CCGC CCTC CTTGGTGATCTTCTCAATGGCGTCC CTCTTGGC
GGTGCCGGCGGTGATGACGTCGTCGACAATCAGGAC CCTCTTGCC
CTTGAGCGAAGCGCCGACGATGTTGCCGCCCTCGCCGTGGTCCTTGGCCTCCTTGCGGTCAAACGAGTAGGAGACGCGG
TCCAGG
TTCTGGGGCGCCAGCTCGCCGAGCTTGATGGTGATGGCGGAGCACAGCGGGATGCCCTTGTAGGCCGGGCCGAAGACGA
TGTCG
AACTCTAGGCCGGCCTTCTCCTGGGCCTCGATGATGGTCTTTGCAAAGGCGGAGGCGATGGCGCCGGCGAGGCGCGCCG
TGTGGA
ATTCGCCCGCGTTGAAGAAGTAGGGGGATATCCGCTTGGACTTGAGCTCGAAGCTGCCAAACTTGAGGACGCCGCCGTC
GATGGC
GGATTTGAGGAAGTCCTGCTTGTAGGCAGGCAGCTGGGAGGTGGTAGCCATTCTGTTGGATTTGGATAGTGTCCTTATT
CTCTGAT
TTGAACAGTAGATCAGGACGAGTGAGAGGGATGCAGAGGTTGGATTGGAGTGGTTGAGCTATAAAATTTAGAGGCGCGC
CGTAT
CGAGTTTTCACATGGAAGTCAAAGCGTACAGTGCGAGCTTGTACGTTGGTCTTAGTATCCCACAAGCTTCTGTCTAGGT
ATGATGA
TGGCTATAAGTCACCCAAGGCAGAACTCATCTTGAAGATTGTCTAGAGTGATTTTACCGCTGATGAAATGACTGGACTC
CCTCCTC
CTGCTCTTATACGAAAAATTGCCTGACTCTGCAAAGGTTGTTTGTCTTGGAAGATGATGTGCCCCCCCATCGCTCTTAT
CTCATACC
CCGCCATCTTTCTAGATTCTCATCTTCAACAAGAGGGGCAATCCATGATCTGCGATCCAGATGTGCTTCTGGCCTCATA
CTCTGCCT
TCAGGTTGATGTTCACTTAATTGGTGACGAATTCAGCTGATTTGCTGCAGTATGCTTTGTGTTGGTTCTTTCCAGGCTT
GTGCCAGC
CATGAGCGCTTTGAGAGCATGTTGTCACCTATAAACTCGAGTAACGGCCACATATTGTTCACTACTTGAATCACATACC
TAATTTT
GATAGAATTGACATGTTTAAAGAGCTGAGGTAGCTTTAATGCCTCTGAAGTATTGTGACACAGCTTCTCACAGAGTGAG
AATGAA
AAGTTGGACTCCCCCTAATGAAGTAAAAGTTTCGTCTCTGAACGGTGAAGAGCATAGATCCGGCATCAACTACCTGGCT
AGACTA
CGACGTCAATTCTGCGGCCTTTTGACCTTTATATATGTCCATTAATGCAATAGATTCTTTTTTTTTTTTTTTTTTTTTT
TTTTTTTTTTT
TTTTTTTTGCCCAATTTCGCAGATCAAAGTGGACGTTATAGCATCATAACTAAGCTCAGTTGCTGAGGGAAGCCGTCTA
CTACCTT
AGCCCATCCATCCAGCTCCATACCTTGATACTTTAGACGTGAAGCAATTCACACTGTACGTCTCGCAGCTCTCCTTCCC
GCTCTTGC
TTCCCCACTGGGGTCCATGGTGCGTGTATCGTCCCCTCCTTAATTAAGGCCATTTAGGCCGTTGCTGGCGTTTTTCCAT
AGGCTCCG
CCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCG
TTTCC
CCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGA
AGCGTGGC
GCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCC
CCCGTTC
AGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGC
AGCCAC
TGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACT
AGAAGG
ACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAA
CCACCG
CTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT
TTCTACG
GGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGCCTGCAGGGCCGATTTTGGTCATGAGATTATCAAAAAGGAT
CTTCAC
CTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAA
TGCTTAA
TCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTAC
GATACGG
GAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAA
ACCAGC
CAGCC GGAAGGGCCGAGC GCAGAAGTGGTCCTGCAACTTTATC CGCCTC CATC CAGTCTATTAATTGTTGCC
GGGAAGCTAGAGT
AAGTAGTTC GCCAGTTAATAGTTTGCGCAAC GTTGTTGC CATTGCTACAGGCATC GTGGTGTCAC GCTC
GTCGTTTGGTATGGC TT
CATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGG
TCCTCCG
ATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGC
CATCCGT
AAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGC
CCGGCGT
CAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACT
CTCAAG
GATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACC
AGCGTTTC
TGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTC
TTCCT
TTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGGCCATTTAGGCCT (Seq ID no 16) Example 2 - Operational p11- and temperature optimum of the PepN 2 from Neosartorya fischeri (TRI031) in gluten hydrolyses
[00300] The liberation of glutamic acid is an important quality parameter in vegetable protein hydrolyses such as soy or gluten hydrolyses. The liberated glutamic acid can be perceived as the so-called "umami-flavor". In order to determine the effect of the initially applied pH in gluten hydrolyses in terms of glutamic acid and glutamine liberation, the initial pH
of a gluten hydrolyses was adjusted just after the liquefaction of the gluten after 15 ¨ 30 min with 1 M HC1. Gluten was treated with Glutaminase (Amano, Japan). In addition, gluten was treated with FoodPro Alkaline Protease, FoodPro PNL as well as PepN 2 (Neosartorya fischeri). After 20 hr, the hydrolysis was terminated employing ultrafiltration (10 kDa cut-off, Sartorius Stedium Biotech, Gottingen, Germany). The permeate was applied for the enzymatic glutamic acid analyses (enzymatic L-glutamic acid analysis kit, Roche, Mannheim, Germany).
As shown in Figure 2, the PepN 2 performed more efficient within the pH range of 7.0 ¨ 9.0 compared to pH
6Ø
of a gluten hydrolyses was adjusted just after the liquefaction of the gluten after 15 ¨ 30 min with 1 M HC1. Gluten was treated with Glutaminase (Amano, Japan). In addition, gluten was treated with FoodPro Alkaline Protease, FoodPro PNL as well as PepN 2 (Neosartorya fischeri). After 20 hr, the hydrolysis was terminated employing ultrafiltration (10 kDa cut-off, Sartorius Stedium Biotech, Gottingen, Germany). The permeate was applied for the enzymatic glutamic acid analyses (enzymatic L-glutamic acid analysis kit, Roche, Mannheim, Germany).
As shown in Figure 2, the PepN 2 performed more efficient within the pH range of 7.0 ¨ 9.0 compared to pH
6Ø
[00301] In order to determine the effect of the applied temperature during the gluten hydrolyses in terms of glutamic acid and glutamine liberation, the initial pH
was adjusted with 1 M HC1 to pH 7.0 just after the liquefaction of the gluten suspension after 15 ¨ 30 min. A
temperature range from 40 ¨ 60 C was applied as shown in Figure 3. Gluten was combined with Glutaminase (Amano, Japan). In addition, gluten was combined FOODPRO Alkaline Protease, FOODPRO PNL as well as PepN 2 (Neosartorya fischeri). After 20 h of hydrolysis, the hydrolysis was terminated employing ultrafiltration (10 kDa cut-off, Sartorius Stedium, Gottingen, Germany). The permeate was applied for the enzymatic glutamic acid analyses (enzymatic L-glutamic acid analysis kit, Roche, Mannheim, Germany). The PepN 2 from Neosartorya fischeri performed most efficient at 60 C.
Example 3 - Comparison of the PepN 2 from Neosartorya fischeri (TRI031) and Aspergillus clavatus (TRI035) in terms of degree of hydrolyses
was adjusted with 1 M HC1 to pH 7.0 just after the liquefaction of the gluten suspension after 15 ¨ 30 min. A
temperature range from 40 ¨ 60 C was applied as shown in Figure 3. Gluten was combined with Glutaminase (Amano, Japan). In addition, gluten was combined FOODPRO Alkaline Protease, FOODPRO PNL as well as PepN 2 (Neosartorya fischeri). After 20 h of hydrolysis, the hydrolysis was terminated employing ultrafiltration (10 kDa cut-off, Sartorius Stedium, Gottingen, Germany). The permeate was applied for the enzymatic glutamic acid analyses (enzymatic L-glutamic acid analysis kit, Roche, Mannheim, Germany). The PepN 2 from Neosartorya fischeri performed most efficient at 60 C.
Example 3 - Comparison of the PepN 2 from Neosartorya fischeri (TRI031) and Aspergillus clavatus (TRI035) in terms of degree of hydrolyses
[00302] The degree of hydrolyses (DH) describes the relative amount of cleaved peptides bounds compared to an acid hydrolyses of the same amount of protein conducted e.g. in 6 M
HCL at ¨120 C for ¨24 h. The higher the DH can therefore be applied to assess the efficiency of amino acid liberation of a general PepN such as a PepN 1 or a PepN 2 type. The cell free culture broth was concentrated (10 kDa cut-off, Sartorius, Gottingen, Germany) and desalted applying a disposal desalting column (PD10, GE, Munchen, Germany) as described by the manufacturer. As substrates were pre-hydrolysed 10% (w/w) Na-caseinate (DMK, Germany), whey protein isolate (WPI; Arla, Viby, Denmark), soy protein isolate (SPI; SUPRO 760, DuPont, Brabrand, Denmark) and gluten suspensions were applied. The pre-hydrolysis was conducted with 1%
(w/wprotein) of FOODPRO Alkaline Protease, 1% w/
(w = = = protein) FOODPRO PNL for the Na-Caseinate, the WPI as well as the SPI suspension. All pre-hydrolyses were conducted at 55 C for 18 h followed by an inactivation step at 95 C for 20 min. Each applied PepN 2 has been inactivated at 95 C for 15 min as a control of the elimination of any carryover of amino acids from the culture broth to the final hydrolyses as well as for the sufficient inactivation of the applied endopeptidases. The hydrolyses were conducted in a 96-well micro titer plate format and consisted of 150 uL of the pre-hydrolyzed protein suspension combined with 50 uL of a PepN 2 stock solution as indicated in Table 1 below. The hydrolyses were conducted at 50 C for 20 hr, subsequently, the hydrolysis were stopped by the addition of 20 uL 2 M TCA
(Trichloroacetic acid, Sigma-Aldrich, Schnelldorf, Germany). The efficiency of the amino acid liberation of the PepN 2 types from Neosartorya fischeri and Aspergillus clavatus can be seen in Table 1 (B) and (A). In particular, for the substrates, Na-caseinate, WPI, and SPI, significantly higher DHs were achieved upon the application of the PepN 2 types from N. fischeri and A.
clavatus. In addition, the achieved DH at a PepN 2 stock solution concentration could be significantly increased. For the gluten hydrolyses, an increased DH was noticed in the protein range from 0.02 ¨ 0.3 mg/mL
PepN 2.
Table 1: Comparison of the degree of hydrolyses of the PepN 2 types from N.
fischeri and A.
clavatus.
A
PepN 2 from Neosartorya fischeri (mg/mL) inactivated Substrate water enzyme at 0.02 0.03 0.04 0.05 0.1 0.15 0.2 0.25 0.3 0.35 the highest conc.
Degree of hydrolysis [%]
Casein 7 8 14 15 16 20 19 21 23 22 24 Gluten 3 4 10 13 15 14 17 18 19 19 21 PepN 2 from Aspergillus clavatus (mg/mL) inactivated enzyme at Substrate water the 0.02 0.03 0.04 0.05 0.1 0.15 0.2 0.25 0.3 0.35 highest conc.
Degree of hydrolysis [%]
Casein 8 8 16 19 19 20 21 23 24 24 24 Gluten 3 4 12 14 15 15 17 18 17 19 19 Example 4 - Gluten hydrolyses applying two endopeptidases in combination with the PepN
2 candidates TRI032, TRI033, TRI034, TRI035, TRI037 and TRI038
HCL at ¨120 C for ¨24 h. The higher the DH can therefore be applied to assess the efficiency of amino acid liberation of a general PepN such as a PepN 1 or a PepN 2 type. The cell free culture broth was concentrated (10 kDa cut-off, Sartorius, Gottingen, Germany) and desalted applying a disposal desalting column (PD10, GE, Munchen, Germany) as described by the manufacturer. As substrates were pre-hydrolysed 10% (w/w) Na-caseinate (DMK, Germany), whey protein isolate (WPI; Arla, Viby, Denmark), soy protein isolate (SPI; SUPRO 760, DuPont, Brabrand, Denmark) and gluten suspensions were applied. The pre-hydrolysis was conducted with 1%
(w/wprotein) of FOODPRO Alkaline Protease, 1% w/
(w = = = protein) FOODPRO PNL for the Na-Caseinate, the WPI as well as the SPI suspension. All pre-hydrolyses were conducted at 55 C for 18 h followed by an inactivation step at 95 C for 20 min. Each applied PepN 2 has been inactivated at 95 C for 15 min as a control of the elimination of any carryover of amino acids from the culture broth to the final hydrolyses as well as for the sufficient inactivation of the applied endopeptidases. The hydrolyses were conducted in a 96-well micro titer plate format and consisted of 150 uL of the pre-hydrolyzed protein suspension combined with 50 uL of a PepN 2 stock solution as indicated in Table 1 below. The hydrolyses were conducted at 50 C for 20 hr, subsequently, the hydrolysis were stopped by the addition of 20 uL 2 M TCA
(Trichloroacetic acid, Sigma-Aldrich, Schnelldorf, Germany). The efficiency of the amino acid liberation of the PepN 2 types from Neosartorya fischeri and Aspergillus clavatus can be seen in Table 1 (B) and (A). In particular, for the substrates, Na-caseinate, WPI, and SPI, significantly higher DHs were achieved upon the application of the PepN 2 types from N. fischeri and A.
clavatus. In addition, the achieved DH at a PepN 2 stock solution concentration could be significantly increased. For the gluten hydrolyses, an increased DH was noticed in the protein range from 0.02 ¨ 0.3 mg/mL
PepN 2.
Table 1: Comparison of the degree of hydrolyses of the PepN 2 types from N.
fischeri and A.
clavatus.
A
PepN 2 from Neosartorya fischeri (mg/mL) inactivated Substrate water enzyme at 0.02 0.03 0.04 0.05 0.1 0.15 0.2 0.25 0.3 0.35 the highest conc.
Degree of hydrolysis [%]
Casein 7 8 14 15 16 20 19 21 23 22 24 Gluten 3 4 10 13 15 14 17 18 19 19 21 PepN 2 from Aspergillus clavatus (mg/mL) inactivated enzyme at Substrate water the 0.02 0.03 0.04 0.05 0.1 0.15 0.2 0.25 0.3 0.35 highest conc.
Degree of hydrolysis [%]
Casein 8 8 16 19 19 20 21 23 24 24 24 Gluten 3 4 12 14 15 15 17 18 17 19 19 Example 4 - Gluten hydrolyses applying two endopeptidases in combination with the PepN
2 candidates TRI032, TRI033, TRI034, TRI035, TRI037 and TRI038
[00303] A
pre-hydrolysed gluten suspension was prepared by addition of FOODPRO
Alkaline Protease (FPAP) and FOODPRO PNL (FPPNL). The hydrolysis was conducted at 55 C. After ¨18 hr, the hydrolysis was terminated by heating to 90 C for 10 min.
After cooling to 50 C, glutaminase was applied to the gluten. Subsequently, 150 !IL of the pre-hydrolyzed gluten was transferred to each well of a 96-well microtiter plate. The glutamic acid liberation was conducted applying 50 !IL (protein concentration: 0.5 mg/mL) of TRI032, TRI033, TRI034, TRI035, TRI037, and TRI038. The hydrolysis was performed for 18 hr and terminated by addition of 20 !IL 2 M TCA. The terminated hydrolyses were filtered (0.22 mm) and further analyzed with the enzymatic glutamic acid analyses kit (Roche, Mannheim, Germany). The liberated glutamic acid concentrations are shown in Figure 4.
Example 5 - Hydrolysis of proline containing peptide
pre-hydrolysed gluten suspension was prepared by addition of FOODPRO
Alkaline Protease (FPAP) and FOODPRO PNL (FPPNL). The hydrolysis was conducted at 55 C. After ¨18 hr, the hydrolysis was terminated by heating to 90 C for 10 min.
After cooling to 50 C, glutaminase was applied to the gluten. Subsequently, 150 !IL of the pre-hydrolyzed gluten was transferred to each well of a 96-well microtiter plate. The glutamic acid liberation was conducted applying 50 !IL (protein concentration: 0.5 mg/mL) of TRI032, TRI033, TRI034, TRI035, TRI037, and TRI038. The hydrolysis was performed for 18 hr and terminated by addition of 20 !IL 2 M TCA. The terminated hydrolyses were filtered (0.22 mm) and further analyzed with the enzymatic glutamic acid analyses kit (Roche, Mannheim, Germany). The liberated glutamic acid concentrations are shown in Figure 4.
Example 5 - Hydrolysis of proline containing peptide
[00304] The peptide WHWLQLKPGQPMY was hydrolysed with TRI031 and TRI035. The peptide (1 mg/mL) was incubated with 1 ug/mL of the aminopeptidase in 20 mM
CPB-Buffer (20 mM Citric acid, 20 mM Phosphate, 20 mM Boric acid) at 55 C. Aliquots (50 L) were stopped with 50 ul 5% TFA at the indicated time points and subjected to LC-MS
analysis.
CPB-Buffer (20 mM Citric acid, 20 mM Phosphate, 20 mM Boric acid) at 55 C. Aliquots (50 L) were stopped with 50 ul 5% TFA at the indicated time points and subjected to LC-MS
analysis.
[00305] The present results show that TRI031 and TRI035 can hydrolyze the peptide WHWLQLKPGQPMY (Figure 5) to KPGQPMY (Figure 6) and further down to QPMY
(Figure 7). That is in contrast to what has been described previously for Aspergillus oryzae PepN 2 (A.M. Blinkovsky et al., Biochimica et Biophysica Acta, 1480 (2000) 171-181, where it was claimed that the Aspergillus oryzae PepN 2 does not hydrolyze any of the X-Pro bonds examined including those of the peptide WHWLQLKPGQPMY.) However, we also find that their enzyme cleaves the peptide down to QPMY (Figure 7).
(Figure 7). That is in contrast to what has been described previously for Aspergillus oryzae PepN 2 (A.M. Blinkovsky et al., Biochimica et Biophysica Acta, 1480 (2000) 171-181, where it was claimed that the Aspergillus oryzae PepN 2 does not hydrolyze any of the X-Pro bonds examined including those of the peptide WHWLQLKPGQPMY.) However, we also find that their enzyme cleaves the peptide down to QPMY (Figure 7).
[00306] Data Acquisition: Capillary LC¨MS/MS analyses were performed using an Agilent 1100 LC system (Agilent Technologies) interfaced to a LTQ Orbitrap Classic hybrid mass spectrometer (Thermo Scientific, Bremen, Germany). Samples were loaded onto a 15 cm Phenomenix Jupiter 4 Proteo 90A, C4 analytical column. Separation was performed at a flow rate of 16 L/min using a 10 min gradient of 0-40% Solvent B H20/CH3CN/ HCOOH
(50/950/0.65 v/v/v) into the IONMAX ion source (Thermo Scientific, San Jose).
The LTQ
Orbitrap Classic instrument was operated in a data-dependent MS/MS mode. The peptide masses were measured by the Orbitrap (MS scans were obtained with a resolution of 60 000 at m/z 400), and up to 2 of the most intense peptide m/z were selected and subjected to fragmentation using CID in the linear ion trap (LTQ). Dynamic exclusion was enabled with a list size of 500 masses, duration of 40 s, and an exclusion mass width of 10 ppm relative to masses on the list.
(50/950/0.65 v/v/v) into the IONMAX ion source (Thermo Scientific, San Jose).
The LTQ
Orbitrap Classic instrument was operated in a data-dependent MS/MS mode. The peptide masses were measured by the Orbitrap (MS scans were obtained with a resolution of 60 000 at m/z 400), and up to 2 of the most intense peptide m/z were selected and subjected to fragmentation using CID in the linear ion trap (LTQ). Dynamic exclusion was enabled with a list size of 500 masses, duration of 40 s, and an exclusion mass width of 10 ppm relative to masses on the list.
[00307] Label free quantification: The RAW files were accessed with the program Skyline 2.6Ø7176(MacLean, B., et at., "Skyline: an open source document editor for creating and analyzing targeted proteomics experiments ", Bioinformatics, 2010, 26(7): p.
966-8) which uses the MS1 intensities to build chromatograms (Schilling, B., et at., "Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline:
application to protein acetylation and phosphorylation", Mot Cell Proteomics, 2012. 11(5): p.
202-14). The precursor isotopic import filter was set to a count of three, (M, M+1, and M+2) at a resolution of 60,000 and the most intense charge state was used. Peptide sequences of the substrate as well as cleavage products were typed into Skyline and intensities were calculated in each sample. Figures were directly copied from the program (See Figure 8).
Example 6: Comparison of the degree of hydrolyses of the PepN 2 from A.
clavatus to the PepN from A. oryzae and L. helveticus ATCC 12046
966-8) which uses the MS1 intensities to build chromatograms (Schilling, B., et at., "Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline:
application to protein acetylation and phosphorylation", Mot Cell Proteomics, 2012. 11(5): p.
202-14). The precursor isotopic import filter was set to a count of three, (M, M+1, and M+2) at a resolution of 60,000 and the most intense charge state was used. Peptide sequences of the substrate as well as cleavage products were typed into Skyline and intensities were calculated in each sample. Figures were directly copied from the program (See Figure 8).
Example 6: Comparison of the degree of hydrolyses of the PepN 2 from A.
clavatus to the PepN from A. oryzae and L. helveticus ATCC 12046
[00308] Enzyme activities of PepN 1 Lactobacillus helveticus ATCC 12046, PepN 1 Aspergillus oryzae and from PepN 2 from Aspergillus clavatus (TRI035) were determined according to Stressler, Eisele et at. (2013, infra). PepN 1 from L. helveticus was expressed and purified as described by Stressler, Eisele et at. (2013, infra).
[00309] PepN 1 from A. oryzae was purified from FLAVOURZYME 500L (Sigma-Aldrich, Schnelldorf, Germany) after desalting on PDio columns equilibrated in 20 mM Bis-Tris, pH 6.5. The sample was purified by anion exchange chromatography, on a Source Q15, XK26/15 (SQ15) column (GE -Lifesciences, USA) and equilibrated with 20 mM
Bis/Tris, pH
6.5 (buffer A). The sample (30 mL) was loaded to the column at a flow rate of 7 mL/min. the column was washed with buffer A and bound proteins were eluted with a linier gradient of 0-0.5 M NaC1 in 20 mM Bis/Tris, pH 6.5 (50 min). During the entire run, fractions of approx. 13 mL
were collected and kept on ice. Fractions with the highest aminopeptidase activity were combined, desalted on PD10 in 20 mM Bis-Tris, pH 6.0 and subjected to a second run on a Poros Q20 HR26/10, XK26/10 column (GE -Lifesciences, USA) equilibrated with 20 mM
Bis/Tris, pH
6.0 (buffer A). The PepN1 sample was loaded to the column at a flow rate of 4 ml/min. the column was washed with buffer A and bound proteins were eluted with a linier gradient of 0-0.25 M NaC1 in 20 mM Bis/Tris, pH 6.0 (30 min). During the entire run, fractions of approx. 8 mL were collected and kept on ice. The purified PepN 1 from A. oryzae was found to have a molecular weight of about 40 kDa according to SDS-PAGE electrophoresis.
Bis/Tris, pH
6.5 (buffer A). The sample (30 mL) was loaded to the column at a flow rate of 7 mL/min. the column was washed with buffer A and bound proteins were eluted with a linier gradient of 0-0.5 M NaC1 in 20 mM Bis/Tris, pH 6.5 (50 min). During the entire run, fractions of approx. 13 mL
were collected and kept on ice. Fractions with the highest aminopeptidase activity were combined, desalted on PD10 in 20 mM Bis-Tris, pH 6.0 and subjected to a second run on a Poros Q20 HR26/10, XK26/10 column (GE -Lifesciences, USA) equilibrated with 20 mM
Bis/Tris, pH
6.0 (buffer A). The PepN1 sample was loaded to the column at a flow rate of 4 ml/min. the column was washed with buffer A and bound proteins were eluted with a linier gradient of 0-0.25 M NaC1 in 20 mM Bis/Tris, pH 6.0 (30 min). During the entire run, fractions of approx. 8 mL were collected and kept on ice. The purified PepN 1 from A. oryzae was found to have a molecular weight of about 40 kDa according to SDS-PAGE electrophoresis.
[00310] The PepN 1 and 2 aminopeptidases were standardized to the activity range of 2.5 ¨
200 nkat*mL1. For protein hydrolyses, 1% (w/wwpi) FOODPRO Alkaline Protease (DuPont, Brabrand, Denmark) and 1% (w/wwpi) FOODPRO PNL (DuPont, Brabrand, Denmark) were added to the whey protein isolate suspension (WPI; LACPRODAN 9224, Arla Ingredients, Viby, Denmark) with an initial pH of 7Ø Following the endopeptidases addition, the WPI was mixed and 200 tL were transferred to each well of a 96-well microtiter plate.
Subsequently, 5 !IL of PepN solution were added as indicated in the Table 2. The hydrolysis was conducted at 50 C without pH control and terminated after 18 hr by addition of 20 tL of 2 M
TCA. Prior to the determination of the DH (Nielsen, Petersen et at., 2001, infra), all samples were filtered 0.22
200 nkat*mL1. For protein hydrolyses, 1% (w/wwpi) FOODPRO Alkaline Protease (DuPont, Brabrand, Denmark) and 1% (w/wwpi) FOODPRO PNL (DuPont, Brabrand, Denmark) were added to the whey protein isolate suspension (WPI; LACPRODAN 9224, Arla Ingredients, Viby, Denmark) with an initial pH of 7Ø Following the endopeptidases addition, the WPI was mixed and 200 tL were transferred to each well of a 96-well microtiter plate.
Subsequently, 5 !IL of PepN solution were added as indicated in the Table 2. The hydrolysis was conducted at 50 C without pH control and terminated after 18 hr by addition of 20 tL of 2 M
TCA. Prior to the determination of the DH (Nielsen, Petersen et at., 2001, infra), all samples were filtered 0.22
[00311] It was found that PepN 2 from A. clavatus achieved high DH than PepN 1 from A.
oryzae at all dosages and higher DH than PepN 1 from L. helveticus at the four highest activity dosages tested. And at the two highest dosages PepN 2 from A. clavatus produced at least 25%
higher DH than the PepN 1 aminopeptidases.
Table 2: Comparison of the degree of hydrolyses of the PepN 2 from A. clavatus to the PepN 1 from L. helveticus and A. oryzae PepN dosage [nkat/mL] in stock solution 0 2,5 5 10 25 50 75 100 200 Degree of hydrolysis [%]
PepN 1: A. oryzae PepN 1: L. helveticus PepN 2: A. c/avatus Example 7: Influence of sodium chloride concentration on glutamic acid liberation in soy and gluten hydrolyses
oryzae at all dosages and higher DH than PepN 1 from L. helveticus at the four highest activity dosages tested. And at the two highest dosages PepN 2 from A. clavatus produced at least 25%
higher DH than the PepN 1 aminopeptidases.
Table 2: Comparison of the degree of hydrolyses of the PepN 2 from A. clavatus to the PepN 1 from L. helveticus and A. oryzae PepN dosage [nkat/mL] in stock solution 0 2,5 5 10 25 50 75 100 200 Degree of hydrolysis [%]
PepN 1: A. oryzae PepN 1: L. helveticus PepN 2: A. c/avatus Example 7: Influence of sodium chloride concentration on glutamic acid liberation in soy and gluten hydrolyses
[00312]
Sodium chloride is frequently applied in the food industry to reduce the microbial contamination risk in protein hydrolyses. Pre-hydrolysed 10% (w/w) soy (SUPRO
760, DuPont, Brabrand, Denmark) and gluten (Sigma-Aldrich, Schnelldorf, Germany) was prepared by addition of 1% (w/wprotem) FOODPRO Alkaline Protease (DuPont, Brabrand, Denmark) and 1% (w/wprotem) FOODPRO PNL (DuPont, Brabrand, Denmark). The hydrolyses were performed at 50 C at pH 7.0 (without pH control) for 18 hr (heat inactivation after 18 hr; 90 C;
min). Subsequently, the hydrolyses were divided in sodium chloride containing (185 mM) and non-salt containing hydrolyses. 150 !IL of each pre-hydrolysed protein suspension was combined with differently standardized Aspergillus clavatus PepN 2 as indicated in Table 3. The hydrolyses were conducted at 50 C without pH control and terminated after 18 hr by addition of 20 tL of 2 M TCA. Prior to the determination of the DH (Nielsen, Petersen et al. 2001, infra), all samples were filtered 0.22 i.tm. As shown in Table 3, the addition of 185 mM NaC1 had no influence on the determined degree of hydrolysis in soy and gluten hydrolyses.
Table 3: Influence of 185 mM NaC1 on the degree of hydrolyses in soy and gluten hydrolyses Protein SOY Gluten Protein concentration [mg/mL]
water 0.2 0.3 0.4 0.5 0.1 0.2 0.25 0.3 0.4 0.5 Degree of hydrolysis 1 1 11 12 12 12 14 16 17 16 17 18 reference 18 185 mM NaCI
Sodium chloride is frequently applied in the food industry to reduce the microbial contamination risk in protein hydrolyses. Pre-hydrolysed 10% (w/w) soy (SUPRO
760, DuPont, Brabrand, Denmark) and gluten (Sigma-Aldrich, Schnelldorf, Germany) was prepared by addition of 1% (w/wprotem) FOODPRO Alkaline Protease (DuPont, Brabrand, Denmark) and 1% (w/wprotem) FOODPRO PNL (DuPont, Brabrand, Denmark). The hydrolyses were performed at 50 C at pH 7.0 (without pH control) for 18 hr (heat inactivation after 18 hr; 90 C;
min). Subsequently, the hydrolyses were divided in sodium chloride containing (185 mM) and non-salt containing hydrolyses. 150 !IL of each pre-hydrolysed protein suspension was combined with differently standardized Aspergillus clavatus PepN 2 as indicated in Table 3. The hydrolyses were conducted at 50 C without pH control and terminated after 18 hr by addition of 20 tL of 2 M TCA. Prior to the determination of the DH (Nielsen, Petersen et al. 2001, infra), all samples were filtered 0.22 i.tm. As shown in Table 3, the addition of 185 mM NaC1 had no influence on the determined degree of hydrolysis in soy and gluten hydrolyses.
Table 3: Influence of 185 mM NaC1 on the degree of hydrolyses in soy and gluten hydrolyses Protein SOY Gluten Protein concentration [mg/mL]
water 0.2 0.3 0.4 0.5 0.1 0.2 0.25 0.3 0.4 0.5 Degree of hydrolysis 1 1 11 12 12 12 14 16 17 16 17 18 reference 18 185 mM NaCI
[00313] Nielsen, P. M., et al. (2001), Journal of Food Science 66(5): 642-646.
[00314] Stressler, T., et at. (2013), PLoS ONE 8(7).
Example 8: Product inhibition of PepN2 TRI031 and TRI035 vs. TRI063 (A.
oryzae)
Example 8: Product inhibition of PepN2 TRI031 and TRI035 vs. TRI063 (A.
oryzae)
[00315] In this assay enzyme activity is determined by hydrolysis of the substrate H-Ala¨
nitroanilide (pNA). The absorbance of released pNA is determined over time at a wavelength of 405 nm using a microtiter plate reader.
nitroanilide (pNA). The absorbance of released pNA is determined over time at a wavelength of 405 nm using a microtiter plate reader.
[00316] As materials buffer 20 mM CPB buffer (20 mM of Na-citrate, Na-phosphate and Na-borate) pH 9.0 was used. Enzyme samples of PepN 2 TRI031 and TRI035 were used. 20 mg H-Ala-pNA substrate (BACHEM, L-1070) was solubilized in 1 mL DMSO (Dimethyl Sulphoxide; Sigma cat# D2650). 96 well plates Costar assay plate 9017 (Corning Inc) were applied and read in a VERSAMAX reader from Molecular Devices. For the inhibition studies mL of a 20 mg/mL solution were made of each of the amino acids: lysine, histidine, leucine, tryptophan, proline, glycine, serine, asparagine, threonine, aspartic acid, glutamic acid.
Tryptophan and proline were dissolved in DMSO. All other amino acids were dissolved in buffer. The final inhibitor mix was made by combining 500 tL of each amino acid solution.
Tryptophan and proline were dissolved in DMSO. All other amino acids were dissolved in buffer. The final inhibitor mix was made by combining 500 tL of each amino acid solution.
[00317] A 96 well plate was placed on ice and 180 tL buffer, 15 tL diluted enzyme and 20 inhibitor mix were added to wells in the plate and mixed. Each plate contained all four enzymes at inhibitor mix concentrations of 0.017, 0.085, 0.128, 0.170, 0.426, 0.851, 1.702, 2.553 mg/mL. The reaction was started by adding 20 tL substrate. Only one substrate concentration was used per plate. After initiating the reaction, the plate was, as quickly as possible, placed in the microplate reader, set to 30 C, and absorbance at 405 nm was measured with 30 sec intervals for 30 min. Six plates were run with one substrate concentrations on each plate (either 0.017, 0.170, 0.426, 0.851, 1.702, 2.553 mg/mL). For determination of the apparent inhibition constant (Ki), data was exported to GraphPad Prism, which fitted Michaelis-Menten curves for each inhibitor concentration and based on this calculated Ki in mg/ml. The unit was converted to mM
by using an average inhibitor molecular weight of 133.9 g/mol. Apparent Ki values for product inhibition and respective standard deviations (SD) for the different enzymes are listed in Table 4.
Table 4 Ki (mM) SD
TRI031 5.67 0.85 TRI035 3.06 0.59 TRI063 (A.
oryzae) 1.20 0.36
by using an average inhibitor molecular weight of 133.9 g/mol. Apparent Ki values for product inhibition and respective standard deviations (SD) for the different enzymes are listed in Table 4.
Table 4 Ki (mM) SD
TRI031 5.67 0.85 TRI035 3.06 0.59 TRI063 (A.
oryzae) 1.20 0.36
[00318] From Table 4 it is seen that PepN 2 TRI031 and TRI035 have the highest apparent Ki values for product inhibition, meaning that they are less inhibited by product compared to TRI063 from A. oryzae.
Example 9: PepN2 and PepN1 activity on Glu-pNA and Gln-pNA substrates
Example 9: PepN2 and PepN1 activity on Glu-pNA and Gln-pNA substrates
[00319] H-Glu¨Nitroanilid (pNA) and H-Gln-Nitroanilid (pNA) were used as substrates and the release of p¨Nitroanilid (pNA) was measured by absorbance at 405 nm.
As buffer 20 mM CPB-Buffer (20 mM Citric acid, 20 mM Na-phosphate, 20 mM Boric acid) pH 9.0 was used. 10 mg H-Glu-pNA and H-Gln-pNA (from BACHEM or Schafer N; Copenhagen, Denmark) were dissolved in 1 ml of DMSO (Dimethyl Sulphoxide from SIGMA; cat #
D2650).
As buffer 20 mM CPB-Buffer (20 mM Citric acid, 20 mM Na-phosphate, 20 mM Boric acid) pH 9.0 was used. 10 mg H-Glu-pNA and H-Gln-pNA (from BACHEM or Schafer N; Copenhagen, Denmark) were dissolved in 1 ml of DMSO (Dimethyl Sulphoxide from SIGMA; cat #
D2650).
[00320] For running the assay 80 tL buffer, 10 tL H-Glu-pNA or H-Gln-pNA
substrate and 10 uL appropriately diluted PepN 2 was incubated in Costar assay plate 9017 (Corning Inc.) at 45 C and measured at 405 nm every 30 sec for 30 min in a VERSAMAX
microplate reader (Molecular Devices) running with SoftMaxPro 5.4.1 software. Activity was determined as the maximal slope of the linear part of the curve determined over 10 time points.
substrate and 10 uL appropriately diluted PepN 2 was incubated in Costar assay plate 9017 (Corning Inc.) at 45 C and measured at 405 nm every 30 sec for 30 min in a VERSAMAX
microplate reader (Molecular Devices) running with SoftMaxPro 5.4.1 software. Activity was determined as the maximal slope of the linear part of the curve determined over 10 time points.
[00321] The assay was run for purified samples of PepN 2 TRI031 and TRI035 as well as purified PepN 1 L. helveticus (Stressler, Eisele et at., supra) and non-purified PepN 1 A. sojae (COROLASE LAP, AB Enzymes) which all had comparable activity on Ala-pNA or Leu-pNA
(run as described for Ala-pNA in Example H, but without inhibitor). As shown in Table 5 the two PepN 2 aminopeptidases showed activity on Glu- and Gln-pNA in contrast to the two PepN
1 enzymes that showed no significant activity. This indicates that PepN 2 aminopeptidases are much more effective in Glu and Gln release than PepN 1 aminopeptidases.
Table 5. Activity of PepN 2 and PepN 1 on Glu-pNA and Gln-pNA substrates Vmax (mU/min) Glu-pNA Gln-pNA
PepN 2 TR1031 50.8 75.9 PepN 2 TR1035 20.1 98.7 Pep N 1 L. helveticus 0.02 0 PepN 1 Corolase LAP 0 0 Example 10 - Hydrolysis of peptide with proline in position 2
(run as described for Ala-pNA in Example H, but without inhibitor). As shown in Table 5 the two PepN 2 aminopeptidases showed activity on Glu- and Gln-pNA in contrast to the two PepN
1 enzymes that showed no significant activity. This indicates that PepN 2 aminopeptidases are much more effective in Glu and Gln release than PepN 1 aminopeptidases.
Table 5. Activity of PepN 2 and PepN 1 on Glu-pNA and Gln-pNA substrates Vmax (mU/min) Glu-pNA Gln-pNA
PepN 2 TR1031 50.8 75.9 PepN 2 TR1035 20.1 98.7 Pep N 1 L. helveticus 0.02 0 PepN 1 Corolase LAP 0 0 Example 10 - Hydrolysis of peptide with proline in position 2
[00322] The peptide library XPAAAR (X being all amino acids except cysteine) was hydrolysed with enzyme samples of TRI032, TRI035, TRI063 (A. oryzae) as well as COROLASE LAP. The peptide library XPAAAR (1 mg/mL) was incubated with 1 ug/mL
of the aminopeptidase in 3 x20 mM CPB-Buffer (20 mM Citric acid, 20 mM Phosphate, 20 mM
Boric acid) at 55 C. 50 aliquots were stopped with 50 ul 5% TFA at the indicated time points and subjected to LC-MS analysis.
of the aminopeptidase in 3 x20 mM CPB-Buffer (20 mM Citric acid, 20 mM Phosphate, 20 mM
Boric acid) at 55 C. 50 aliquots were stopped with 50 ul 5% TFA at the indicated time points and subjected to LC-MS analysis.
[00323] Data Acquisition: Capillary LC¨MS/MS analyses were performed using an Agilent 1100 LC system (Agilent Technologies) interfaced to a LTQ Orbitrap Classic hybrid mass spectrometer (Thermo Scientific, Bremen, Germany). Samples were loaded onto a 15 cm Phenomenex Jupiter 4 Proteo 90A, C4 analytical column. Separation was performed at a flow rate of 16 L/min using a 10 min gradient of 0-40% Solvent B H20/CH3CN/ HCOOH
(50/950/0.65 v/v/v) into the IONMAX ion source (Thermo Scientific, San Jose).
The LTQ
Orbitrap Classic instrument was operated in a data-dependent MS/MS mode. The peptide masses were measured by the Orbitrap (MS scans were obtained with a resolution of 60 000 at m/z 400), and up to 2 of the most intense peptide m/z were selected and subjected to fragmentation using CID in the linear ion trap (LTQ). Dynamic exclusion was enabled with a list size of 500 masses, duration of 40 s, and an exclusion mass width of 10 ppm relative to masses on the list.
(50/950/0.65 v/v/v) into the IONMAX ion source (Thermo Scientific, San Jose).
The LTQ
Orbitrap Classic instrument was operated in a data-dependent MS/MS mode. The peptide masses were measured by the Orbitrap (MS scans were obtained with a resolution of 60 000 at m/z 400), and up to 2 of the most intense peptide m/z were selected and subjected to fragmentation using CID in the linear ion trap (LTQ). Dynamic exclusion was enabled with a list size of 500 masses, duration of 40 s, and an exclusion mass width of 10 ppm relative to masses on the list.
[00324] Label free quantification: The RAW files were accessed with the program Skyline 2.6Ø7176 (MacLean, B., et at., supra) which uses the MS1 intensities to build chromatograms (Schilling, B., et at., supra). The precursor isotopic import filter was set to a count of three, (M, M+1, and M+2) at a resolution of 60,000 and the most intense charge state was used. Peptide sequences of the substrate as well as cleavage products were typed into Skyline and intensities were calculated in each sample. Figure 10 was generated on the basis the sum of the peak areas of the three isotopes (M, M+1, and M+2). A substrate standard curve (with serial 2 fold increases in concentration from Std 3.125 to Std 50) was included and shows a linear relationship between substrate concentration and peak area.
[00325] LC-MS results (Figure 10) show that TRI032 and TRI035 can hydrolyze the peptide TPAAAR over time to less than half the concentration within 2 hr incubation, whereas TRI063 (A. oryzae) and COROLASE LAP show no hydrolysis of TPAAAR within 12 hr.
[00326] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
Claims (29)
1. An isolated polypeptide having aminopeptidase activity, selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 80%
identity with the amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ
ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8; and (b) a fragment of (a), wherein the fragment has aminopeptidase activity.
(a) a polypeptide having an amino acid sequence which has at least 80%
identity with the amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ
ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8; and (b) a fragment of (a), wherein the fragment has aminopeptidase activity.
2. The polypeptide of claim 1, comprising an amino acid sequence which has at least 90%
identity with the amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ
ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO:
8.
identity with the amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ
ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO:
8.
3. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO: 1, or SEQ ID
NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID
NO: 7, or SEQ ID NO: 8 or a fragment thereof.
NO: 1, or SEQ ID
NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID
NO: 7, or SEQ ID NO: 8 or a fragment thereof.
4. The polypeptide of claim 3, comprising the amino acid sequence of SEQ ID
NO: 1, or SEQ ID
NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID
NO: 7, or SEQ ID NO: 8.
NO: 1, or SEQ ID
NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID
NO: 7, or SEQ ID NO: 8.
5. The polypeptide of any of claims 1 to 4, wherein said fragment comprises any one of SEQ ID
NOs: 18 to 25.
NOs: 18 to 25.
6. An isolated polypeptide having aminopeptidase activity which has a predicted mature sequence with more than 13 residues on the N-terminal side of the conserved residue IN in position 67 as shown in the sequence alignment of Figure 9, or a fragment thereof, wherein the fragment has aminopeptidase activity.
7. An isolated polypeptide that has aminopeptidase activity, and which has decreased product inhibition compared to the aminopeptidase of SEQ ID NO:17.
8. The isolated polypeptide of claim 7 wherein said polypeptide is as defined in any one of claims 1 to 6.
9. An isolated polypeptide that has aminopeptidase activity, and which is capable of hydrolyzing a polypeptide with a proline residue in position 2, as numbered from the N-terminus.
10. The isolated polypeptide of claim 9, which is capable of hydrolyzing a polypeptide with a proline residue in position 2, as numbered from the N-terminus, to less than half the starting concentration within 2 hours of incubation.
11. The isolated polypeptide of claim 9 or 10, wherein said isolated polypeptide is as defined in any one of claims 1 to 8.
12. An isolated nucleic acid sequence comprising a nucleic acid sequence which encodes the polypeptide of any of claims 1 to 8.
13. A nucleic acid construct comprising the nucleic acid sequence of claim 12 operably linked to one or more control sequences which direct the production of the polypeptide in a suitable expression host.
14. A recombinant expression vector comprising the nucleic acid construct of claim 13, a promoter, and transcriptional and translational stop signals.
15. A recombinant host cell comprising the nucleic acid construct of claim 13.
16. The recombinant host cell of claim 15, wherein the host cell is a Trichoderma cell, preferably a Trichoderma reesei cell.
17. A method for producing the polypeptide of any of claims 1 to 11 comprising (a) cultivating a strain to produce a supernatant comprising the polypeptide; and (b) recovering the polypeptide.
18. A method for producing the polypeptide of any of claims 1 to 11 comprising (a) cultivating a host cell of claims 15 or 16 under conditions suitable for production of the polypeptide; and (b) recovering the polypeptide.
19. A method for producing the polypeptide of any of claims 1 to 11 comprising (a) cultivating a homologous recombinant cell, having incorporated therein a new transcription unit comprising a regulatory sequence, an exon, and/or a splice donor site operably linked to a second exon of an endogenous nucleic acid sequence encoding the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
20. The method of claim 19, wherein the host cell is a Trichoderma cell, preferably a Trichoderma reesei cell.
21. A method for the production of a protein hydrolysate comprising subjecting a proteinaceous substrate to a polypeptide of any of claims 1 to 11.
22. The method of claim 21, further comprising subjecting said proteinaceous substrate to an endopeptidase.
23. The method of claim 21 or 22, wherein the hydrolysate is enriched in Leu, Gly, Glu, Ser, Asp, Asn, Pro, Cys, Ala, and/or Gln.
24. The method of claim 23, wherein the hydrolysate is enriched in Glu and/or Gln.
25. A method for obtaining from a proteinaceous substrate a protein hydrolysate enriched in free glutamic acid and/or peptide bound glutamic acid residues, comprising subjecting the substrate to a deamidation process and a polypeptide of any of claims 1 to 11.
26. The method of claim 25, further comprising subjecting the substrate to one or more unspecific acting endo- and/or exo-peptidase enzymes.
27. A composition comprising a polypeptide of any of claims 1 to 11 and a suitable carrier.
28. A method of producing malted cereal comprising:
a) providing a cereal grain, b) adding to the cereal an effective amount of the polypeptide of any of claims 1 to 11 during a malting process; and c) obtaining a malted cereal.
a) providing a cereal grain, b) adding to the cereal an effective amount of the polypeptide of any of claims 1 to 11 during a malting process; and c) obtaining a malted cereal.
29. A method for the production of free amino nitrogen (FAN) during brewing comprising adding an effective amount of the polypeptide of any of claims 1 to 11 during a brewing process.
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US20180291360A1 (en) | 2018-10-11 |
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BR112017028035A2 (en) | 2018-08-28 |
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CN107849595A (en) | 2018-03-27 |
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